Circular RNA for translation in eukaryotic cells

ABSTRACT

Methods and constructs for engineering circular RNA are disclosed. In some embodiments, the methods and constructs comprise a vector for making circular RNA, the vector comprising the following elements operably connected to each other and arranged in the following sequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′ splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3′ spacer sequence, f.) a 5′ Group I intron fragment containing a 5′ splice site dinucleotide, and g.) a 3′ homology arm, the vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. Methods for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector are also disclosed.

RELATED APPLICATION(S)

This application is a Divisional of U.S. application Ser. No.17/191,697, filed Mar. 3, 2021, with is a Continuation of U.S.application Ser. No. 16/432,177, filed Jun. 5, 2019, which claims thebenefit of U.S. Provisional Application No. 62/851,548, filed on May 22,2019, U.S. Provisional Application No. 62/791,028, filed on Jan. 10,2019 and U.S. Provisional Application No. 62/681,617, filed on Jun. 6,2018. The entire teachings of the above applications are incorporatedherein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under W32P4Q-13-1-0011from Defense Advanced Research Projects Agency and under 5R01HL125428from National Institutes of Health. The government has certain rights inthe invention.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

-   -   a) File name: 00502311017_Sequence_Listing.txt; created Jun. 12,        2021, 56 KB in size.

BACKGROUND

Messenger RNA (mRNA) has broad potential for a range of therapeutic andengineering applications. However, one fundamental limitation to its useis its relatively short half-life in biological systems. Thus, there isa need to extend the duration of protein expression from full-length RNAmessages.

SUMMARY

In certain aspects, provided herein is a vector for making circular RNA(circRNA).

In some embodiments, the vector comprises the following elementsoperably connected to each other and, in some embodiments, arranged inthe following sequence: a.) a 5′ homology arm, b.) a 3′ group I intronfragment containing a 3′ splice site dinucleotide, c.) a protein codingor noncoding region, d.) a 5′ group I intron fragment containing a 5′splice site dinucleotide, and e.) a 3′ homology arm. In certainembodiments said vector allows production of a circular RNA that istranslatable and/or biologically active inside eukaryotic cells. In someembodiments, the biologically active RNA is, for example, an miRNAsponge, or long non-coding RNA.

In some embodiments, said vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.)optionally, an internal ribosome entry site (IRES), e.) a protein codingor noncoding region, f.) optionally, a 3′ spacer sequence, g.) a 5′group I intron fragment containing a 5′ splice site dinucleotide, andh.) a 3′ homology arm. In certain embodiments, said vector allowsproduction of a circular RNA that is translatable and/or biologicallyactive inside eukaryotic cells.

In some embodiments, the vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internalribosome entry site (IRES), e.) a protein coding or noncoding region,f.) a 5′ group I intron fragment containing a 5′ splice sitedinucleotide, and g.) a 3′ homology arm. In some embodiments, saidvector allows production of a circular RNA that is translatable and/orbiologically active inside eukaryotic cells.

In some embodiments, the vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein codingor noncoding region, e.) a 3′ spacer sequence, f.) a 5′ group I intronfragment containing a 5′ splice site dinucleotide, and g.) a 3′ homologyarm. In some embodiments, said vector allows production of a circularRNA that is translatable and/or biologically active inside eukaryoticcells.

In some embodiments, said vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) an internal ribosome entry site (IRES),d.) a protein coding or noncoding region, e.) a 3′ spacer sequence, f.)a 5′ group I intron fragment containing a 5′ splice site dinucleotide,and g.) a 3′ homology arm. In some embodiments, said vector allowsproduction of a circular RNA that is translatable and/or biologicallyactive inside eukaryotic cells.

In some embodiments, said vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) a protein coding or noncoding region, d.)a 3′ spacer sequence, e.) a 5′ group I intron fragment containing a 5′splice site dinucleotide, and f) a 3′ homology arm. In some embodiments,said vector allows production of a circular RNA that is translatableand/or biologically active inside eukaryotic cells.

In some embodiments, said vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) a 5′ spacer sequence, d.) a protein codingor noncoding region, e.) a 5′ group I intron fragment containing a 5′splice site dinucleotide, and f) a 3′ homology arm. In some embodiments,said vector allows production of a circular RNA that is translatableand/or biologically active inside eukaryotic cells.

In some embodiments, said vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) an internal ribosome entry site (IRES),d.) a protein coding or noncoding region, e.) a 5′ group I intronfragment containing a 5′ splice site dinucleotide, and f.) a 3′ homologyarm. In some embodiments, said vector allows production of a circularRNA that is translatable and/or biologically active inside eukaryoticcells.

In some embodiments, the vector comprises the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) a 5′ spacer sequence, d.) an internalribosome entry site (IRES), e.) a protein coding or noncoding region,f.) a 3′ spacer sequence, g.) a 5′ group I intron fragment containing a5′ splice site dinucleotide, and h.) a 3′ homology arm. In someembodiments, said vector allowing production of a circular RNA that istranslatable and/or biologically active inside eukaryotic cells.

In one embodiment, the 3′ group I intron fragment and/or the 5′ group Iintron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu geneor T4 phage Td gene.

In one embodiment, the 3′ group I intron fragment and/or the 5′ group Iintron fragment is from a Cyanobacterium Anabaena sp. pre-tRNA-Leu gene.

In another embodiment, if present, the IRES sequence is an IRES sequenceof Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitisvirus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padivirus, Reticuloendotheliosis virus, fuman poliovirus 1, Plautia staliintestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodiscacoagulata virus-1, Human Immunodeficiency Virus type 1, Homalodiscacoagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis Avirus, Hepatitis GB virus, foot and mouth disease virus, Humanenterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-likevirus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifertobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1,Black Queen Cell Virus, Aphid lethal paralysis virus, Avianencephalomyelitis virus, Acute bee paralysis virus, Hibiscus chloroticringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1,Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, HumanBAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G,Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx,Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3,Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, MouseUtrA, Human VEGF-A, Human XIAP, Salivirus, Cosavirus, Parechovirus,Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Humanc-src, Human FGF-1, Simian picomavirus, Turnip crinkle virus, an aptamerto eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1/2). In yetanother embodiment, the IRES is an IRES sequence of Coxsackievirus B3(CVB3). In a further embodiment, the IRES is an IRES sequence ofEncephalomyocarditis virus.

In one embodiment, the protein coding region encodes a protein ofeukaryotic or prokaryotic origin. In another embodiment, the proteincoding region encodes human protein or non-human protein. In someembodiments, the protein coding region encodes one or more antibodies.For example, in some embodiments, the protein coding region encodeshuman antibodies. In one embodiment, the protein coding region encodes aprotein selected from hFIX, SP-B, VEGF-A, human methylmalonyl-CoA mutase(hMUT), CFTR, cancer self-antigens, and additional gene editing enzymeslike Cpfl, zinc finger nucleases (ZFNs) and transcription activator-likeeffector nucleases (TALENs). In another embodiment, the protein codingregion encodes a protein for therapeutic use. In one embodiment, thehuman antibody encoded by the protein coding region is an anti-HIVantibody. In one embodiment, the antibody encoded by the protein codingregion is a bispecific antibody. In one embodiment, the bispecificantibody is specific for CD19 and CD22. In another embodiment, thebispecific antibody is specific for CD3 and CLDN6. In one embodiment,the protein coding region encodes a protein for diagnostic use. In oneembodiment, the protein coding region encodes Gaussia luciferase (Gluc),Firefly luciferase (Flue), enhanced green fluorescent protein (eGFP),human erythropoietin (hEPO), or Cas9 endonuclease.

In one embodiment, the 5′ homology arm is about 5-50 nucleotides inlength. In another embodiment, the 5′ homology arm is about 9-19nucleotides in length. In some embodiments, the 5′ homology arm is atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19nucleotides in length. In some embodiments, the 5′ homology arm is nomore than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In someembodiments, the 5′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or 19 nucleotides in length.

In one embodiment, the 3′ homology arm is about 5-50 nucleotides inlength. In another embodiment, the 3′ homology arm is about 9-19nucleotides in length. In some embodiments, the 3′ homology arm is atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19nucleotides in length. In some embodiments, the 3′ homology arm is nomore than 50, 45, 40, 35, 30, 25 or 20 nucleotides in length. In someembodiments, the 3′ homology arm is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or 19 nucleotides in length.

In one embodiment, the 5′ spacer sequence is at least 10 nucleotides inlength. In another embodiment, the 5′ spacer sequence is at least 15nucleotides in length. In a further embodiment, the 5′ spacer sequenceis at least 30 nucleotides in length. In some embodiments, the 5′ spacersequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25 or 30 nucleotides in length. In some embodiments, the 5′ spacersequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30nucleotides in length. In some embodiments the 5′ spacer sequence isbetween 20 and 50 nucleotides in length. In certain embodiments, the 5′spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In oneembodiment, the 5′ spacer sequence is a polyA sequence. In anotherembodiment, the 5′ spacer sequence is a polyA-C sequence.

In one embodiment, the 3′ spacer sequence is at least 10 nucleotides inlength. In another embodiment, the 3′ spacer sequence is at least 15nucleotides in length. In a further embodiment, the 3′ spacer sequenceis at least 30 nucleotides in length. In some embodiments, the 3′ spacersequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25 or 30 nucleotides in length. In some embodiments, the 3′ spacersequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30nucleotides in length. In some embodiments the 3′ spacer sequence isbetween 20 and 50 nucleotides in length. In certain embodiments, the 3′spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In oneembodiment, the 3′ spacer sequence is a polyA sequence. In anotherembodiment, the 5′ spacer sequence is a polyA-C sequence.

In one embodiment, the vector further comprises an RNA polymerasepromoter. In another embodiment, the RNA polymerase promoter is a T7virus RNA polymerase promoter, T6 virus RNA polymerase promoter, SP6virus RNA polymerase promoter, T3 virus RNA polymerase promoter, or T4virus RNA polymerase promoter.

In one embodiment, the vector is used to transcribe circular RNA withthe size range of about 500 to about 10,000 nucleotides. In someembodiments, the circular RNA is at least 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000nucleotides in size. In some embodiments, the circular RNA is no morethan 10,000, 9,000, 8,000, 7,000, 6,000, 5,000 or 4,000 nucleotides insize.

In another embodiment, the IRES is an IRES sequence from CoxsackievirusB3 (CVB3), the protein coding region encodes Guassia luciferase (Gluc)and the spacer sequences are polyA-C.

In some embodiments, the IRES, if present, is at least about 50nucleotides in length. In one embodiment, the vector comprises an IRESthat comprises a natural sequence. In one embodiment, the vectorcomprises an IRES that comprises a synthetic sequence.

In one embodiment, the invention is directed to a vector for makingcircular RNA, said vector comprising the following elements operablyconnected to each other and arranged in the following sequence: a.) a 5′homology arm, b.) a 3′ group I intron fragment containing a 3′ splicesite dinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosomeentry site (IRES), e.) a protein coding or noncoding region, f) a 5′group I intron fragment containing a 5′ splice site dinucleotide, andg.) a 3′ homology arm. In some embodiments, said vector allowsproduction of a circular RNA that is translatable and/or biologicallyactive inside eukaryotic cells.

In one embodiment, the invention is directed to a vector for makingcircular RNA, said vector comprising the following elements operablyconnected to each other and arranged in the following sequence: a.) a 5′homology arm, b.) a 3′ group I intron fragment containing a 3′ splicesite dinucleotide, c.) an internal ribosome entry site (IRES), d.) aprotein coding or noncoding region, e.) a spacer (e.g., second spacer)sequence, f.) a 5′ group I intron fragment containing a 5′ splice sitedinucleotide, and g.) a 3′ homology arm. In some embodiments, saidvector allows production of a circular RNA that is translatable and/orbiologically active inside cells, e.g., eukaryotic cells.

In certain embodiments, the vectors provided herein do not comprise amulti cloning site (MCS).

In one embodiment, the invention is directed to a circular RNA. Incertain embodiments, the circular RNA is a circular RNA produced by avector provided herein. In some embodiments, the circular RNA comprises,in the following sequence: a.) a 5′ spacer sequence, b.) an internalribosome entry site (IRES), c.) a protein coding or noncoding region,and d.) a 3′ spacer sequence. In some embodiments, the circular RNAfurther comprises the portion of the 3′ group I intron fragment that is3′ of the 3′ splice site dinucleotide. In some embodiments, the circularRNA further comprises the portion of the 5′ group I intron fragment thatis 5′ of the 5′ splice site dinucleotide. In some embodiments, thecircular RNA is at least 500, 600, 700, 800, 900, 1000, 1500, 2000,2500, 3000, 3500, 4000 or 4500 nucleotides. In one embodiment, thecircular RNA is at least about 10 nt. In one embodiment, the circularRNA is about 500 nt or less than 500 nt. In one embodiment, the circularRNA is at least about 1 kb. The circular RNA can be unmodified,partially modified or completely modified. In one embodiment, thecircular RNA contains at least one nucleoside modification. In oneembodiment, up to 100% of the nucleosides of the circular RNA aremodified. In one embodiment, at least one nucleoside modification is auridine modification or an adenosine modification. In one embodiment, atleast one nucleoside modification is selected from N6-methyladenosine(m6A), pseudouridine (ψ), N¹-methylpseudouridine (m1ψ), and5-methoxyuridine (5moU). In one embodiment, the precursor RNA ismodified with methylpseudouridine (m1ψ).

In another embodiment, the invention is directed to a method ofexpressing protein in a cell, said method comprising transfecting thecircular RNA into the cell. In one embodiment, the method comprisestransfecting using lipofection or electroporation. In anotherembodiment, the circular RNA is transfected into a cell using ananocarrier. In yet another embodiment, the nanocarrier is a lipid,polymer or a lipo-polymeric hybrid. In one embodiment, the circular RNAcomprises coxsackievirus B3 IRES.

In one embodiment, the invention is directed to a method of purifyingcircular RNA, comprising running the RNA through a size-exclusion columnin tris-EDTA or citrate buffer in a high performance liquidchromatography (HPLC) system. In another embodiment, the RNA is runthrough the size-exclusion column in tris-EDTA or citrate buffer at pHin the range of about 4-7 at a flow rate of about 0.01-5 mL/minute. Inone embodiment, the HPLC removes one or more of: intron fragments,nicked linear RNA, linear and circular concatenations, and impuritiesresulting from the in vitro transcription and splicing reactions.

In one embodiment, provided herein is a precursor RNA. In certainembodiments, the precursor RNA is a circular RNA produced by in vitrotranscription of a vector provided herein. In some embodiments, theprecursor RNA comprises, in the following sequence, a.) a 5′ homologyarm, b.) a 3′ group I intron fragment containing a 3′ splice sitedinucleotide, c.) a 5′ spacer sequence, d.) an internal ribosome entrysite (IRES), e.) a protein coding or noncoding region, f.) a 3′ spacersequence, f.) a 5′ group I intron fragment containing a 5′ splice sitedinucleotide, and g.) a 3′ homology arm. The precursor RNA can beunmodified, partially modified or completely modified. In oneembodiment, the precursor RNA contains at least one nucleosidemodification. In one embodiment, up to 100% of the nucleosides of theprecursor RNA are modified. In one embodiment, at least one nucleosidemodification is a uridine modification or an adenosine modification. Inone embodiment, at least one nucleoside modification is selected fromN6-methyladenosine (m6A), pseudouridine (ψ), N¹-methylpseudouridine(m1ψ), and 5-methoxyuridine (5moU). In one embodiment, the precursor RNAis modified with methylpseudouridine (m1ψ).

In another embodiment, the invention is directed to a method ofpurifying circular RNA, said method comprising: running circular RNA(e.g., circular RNA provided herein) through a size-exclusion column intris-EDTA or citrate buffer in a high-performance liquid chromatography(HPLC) system, and treating the circular RNA with phosphatase afterrunning the circular RNA through the size-exclusion column, therebyproducing purified circular RNA. In one embodiment, the phosphatasetreatment is followed by RNase R treatment. In one embodiment, thepurified circular RNA is formulated into nanoparticles. In oneembodiment, the circular RNA is run through the size-exclusion column ata pH in the range of about 4-8. In one embodiment, the circular RNA isrun through the size-exclusion column at a flow rate of about 0.01-5.0mL/minute.

In some embodiments, the HPLC as utilized in the methods herein caninclude an aqueous buffer that includes a salt, such as phosphatebuffer, having a pH of between about 4 and about 7.5.

In yet another embodiment, the invention is directed to a method ofmaking circular RNA from precursor RNA, said method comprising using avector provided herein. In some embodiments, the method comprises a.)synthesizing precursor RNA by in vitro transcription of the vector, andb.) incubating the precursor RNA in the presence of magnesium ions andquanosine nucleotide or nucleoside at a temperature at which RNAcircularization occurs (e.g., between 20° C. and 60° C.). In someembodiments the vector comprises the following elements operablyconnected to each other and arranged in the following sequence: a) a 5′homology arm, b) a 3′ group I intron fragment containing a 3′ splicesite dinucleotide, c) a 5′ spacer sequence, d) a protein coding ornoncoding region, e) a 3′ spacer sequence, f) a 5′ group I intronfragment containing a 5′ splice site dinucleotide, and g) a 3′ homologyarm, said vector allowing production of a circular RNA that istranslatable inside eukaryotic cells. In one embodiment, the methodfurther comprises an internal ribosome entry site (IRES) between the 5′spacer sequence and the protein coding region.

In one embodiment, the invention is directed to a method for makingcircular RNA from precursor RNA generated by in vitro transcription of avector provided herein. In some embodiments, the method includesincubating the precursor RNA in the presence of magnesium ions andquanosine nucleotide or nucleoside at a temperature at which RNAcircularization occurs (e.g., between 20° C. and 60° C.). In someembodiments, the nucleosides of the precursor RNA are unmodified. Theprecursor RNA can be unmodified, partially modified or completelymodified. In one embodiment, the precursor RNA can be naturallyoccurring. In one embodiment, the precursor RNA contains at least onenucleoside modification. In one embodiment, up to 100% of thenucleosides of the precursor RNA are modified. In one embodiment, atleast one nucleoside modification is a uridine modification or anadenosine modification. In one embodiment, at least one nucleosidemodification is selected from N6-methyladenosine (m6A), pseudouridine(ψ), N¹-methylpseudouridine (m1ψ), and 5-methoxyuridine (5moU). In oneembodiment, the precursor RNA is modified with methylpseudouridine(m1ψ).

In one embodiment, the invention is directed to a circular RNA producedby a vector and/or a method disclosed herein. In one embodiment, theinvention is directed to a composition, e.g., a pharmaceuticalcomposition, comprising a circular RNA provided herein (e.g., a circularRNA produced by a vector, precursor RNA and/or a method disclosedherein).

In one embodiment, the invention is directed to a method of expressingprotein in a cell, said method comprising transfecting a circular RNAprovided herein into the cell.

As used herein, “precursor RNA” refers to a linear RNA molecule createdby in vitro transcription (e.g., from a vector provided herein). Thisprecursor RNA molecule contains the entirety of the circRNA sequence,plus splicing sequences (intron fragments and homology arms) necessaryto circularize the RNA. These splicing sequences (intron fragments andhomology arms) are removed from the precursor RNA duringcircularization, yielding circRNA plus two intron/homology arm linearRNA fragments. Precursor RNA can be unmodified, partially modified orcompletely modified. In one embodiment, the precursor RNA contains onlynaturally occurring nucleotides.

In one embodiment, the invention is directed to a method of makingcircular RNA with enhanced translation efficiency, said methodcomprising incorporating artificial nucleosides into a precursor RNAduring transcription of a vector encoding the precursor RNA andcircularizing the precursor RNA to form the circular RNA.

In another embodiment, the invention is directed to a method of makingcircular RNA with enhanced protein expression stability, said methodcomprising incorporating artificial nucleosides into a precursor RNAduring transcription of a vector encoding the precursor RNA andcircularizing the precursor RNA to form the circular RNA.

In yet another embodiment, the invention is directed to a method ofmaking circular RNA with reduced immunogenicity said method comprisingincorporating artificial nucleosides into a precursor RNA duringtranscription of a vector encoding the precursor RNA and circularizingthe precursor RNA to form the circular RNA.

In some embodiments, a vector provided herein can be used to transcribea precursor RNA that will self-splice into a circRNA under the rightconditions (e.g., conditions provided herein). In one embodiment, thelength of this circRNA is between about 200 and about 10,000 nucleotideslong.

In one embodiment, the vectors provided herein comprise an RNApolymerase promoter upstream of the region that encodes the precursorRNA (e.g., upstream of the 5′ homology arm). In some embodiments, thepromoter can be recognized by the T7 phage RNA polymerase enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing will be apparent from the following more particulardescription of example embodiments, as illustrated in the accompanyingdrawings in which like reference characters refer to the same partsthroughout the different views. The drawings are not necessarily toscale, emphasis instead being placed upon illustrating embodiments.

FIG. 1A is a schematic diagram showing an example of a permutedintron-exon construct design and mechanism of splicing. The group Icatalytic intron of the T4 phage Td gene is bisected in such a way as topreserve structural elements critical for ribozyme folding. Exonfragment 2 (E2) is ligated upstream of exon fragment 1 (E1), and acoding region approximately 1.1 kb in length is inserted at theexon-exon junction. During splicing, the 3′ hydroxyl group of aguanosine nucleotide engages in a transesterification reaction at the 5′splice site. The 5′ intron fragment is excised, and the freed hydroxylgroup at the end of the intermediate engages in a secondtransesterification at the 3′ splice site, resulting in circularizationof the intervening region and excision of the 3′ intron.

FIG. 1B shows RNA Fold predictions of precursor RNA secondary structurefor homology arm design. Colors denote base pairing probability, withred indicating higher probability. Without homology arms, no basepairing is predicted to occur between the ends of the precursormolecule. The arrows point to the added homology arms.

FIG. 1C shows an agarose gel demonstrating the effect of homology armson splicing. Putative circRNA runs at a higher molecular weight thanheavier precursor RNA, as indicated. (−): no homology arms. Weak: weakhomology arms, 9 nt. Strong: strong homology arms, 19 nt.

FIG. 1D shows agarose gel confirmation of precursor RNA circularization.C: precursor RNA (with strong homology arms) subjected tocircularization conditions. C+R: Lane C, digested with RNase R. C+R+H:Lane C+R, digested with oligonucleotide-guided RNase H. U: precursor RNAnot subjected to circularization conditions. U+H: Lane U, digested witholigonucleotide-guided RNase H.

FIG. 1E shows sanger sequencing output of RT-PCR across the splicejunction of the sample depicted in lane C+R from FIG. 1D.

FIG. 1F shows RNAFold predictions of precursor RNA secondary structurein the context of designed spacers. Secondary structures potentiallyimportant for ribozyme function are identified by black arrows.

FIG. 1G shows an agarose gel demonstrating the effect of spacers onsplicing. (−): no spacer. D: disruptive spacer. P1: permissive spacer 1.P2: permissive spacer 2.

FIG. 1H shows RNAFold predictions of precursor RNA secondary structurefor internal homology region design. Lack of significant internalhomology (Anabaena 1.0) and introduced internal homology (Anabaena 2.0)indicated by black arrows. ‘Splicing bubble’ indicated as the regionbetween homology arms and internal homology regions that contains thesplicing ribozyme.

FIG. 1I shows an agarose gel demonstrating the effect of internalhomology on splicing.

FIG. 1J shows an agarose gel comparing the optimized T4 phage splicingreaction to the optimized Anabaena splicing reaction. Anabaena intronhalves are of roughly equal lengths, and are less likely to remainassociated after splicing in comparison to the T4 phage intron halvesdespite stronger homology arms.

FIG. 2A is a schematic diagram showing elements of an example engineeredself-splicing precursor RNA design. Evaluation of circularizationefficacy and translation for a range of protein-coding circRNAsgenerated from de-novo engineered precursor RNA.

FIG. 2B shows an agarose gel of precursor RNA containing an EMCV IRESand a variable insert including Gaussia luciferase (GLuc), humanerythropoietin (hEpo), EGFP, Firefly luciferase (FLuc), or Cas9 codingregions after circularization and recircularization (R−). CircRNA wasenriched by RNase R degradation (R+).

FIG. 2C is a bar graph showing approximate circRNA yields from treatmentof 20 μg of splicing reaction with RNase R, as assessed byspectrophotometry (data presented as mean+SD, n=3).

FIG. 2D is a bar graph showing luminescence in the supernatant of HEK293cells 24 hours after transfection with circRNA coding for GLuc (datapresented as mean+SD, n=4, *p<0.05).

FIG. 2E is a bar graph showing the expression of human erythropoietin inthe supernatant of HEK293 cells 24 hours after transfection with circRNAcoding for hEpo (data presented as mean+SD, n=4, *p<0.05).

FIG. 2F shows GFP fluorescence in HEK293 cells 24 hours aftertransfection with circRNA coding for EGFP.

FIG. 2G is a bar graph showing luminescence in the lysate of HEK293cells 24 hours after transfection with circRNA coding for FLuc (datapresented as mean+SD, n=4, *p<0.05).

FIG. 2H is a graph showing FACS analysis demonstrating GFP ablation inHEK293-EF1a-GFP cells 4 days after transfection with sgGFP alone(circCas9−) indicated by the appearance of a GFP-negative cellpopulation.

FIG. 2I is a graph showing FACS analysis demonstrating GFP ablation inHEK293-EF1a-GFP cells 4 days after transfection with circRNA coding forCas9 (circCas9+), indicated by the appearance of a GFP-negative cellpopulation.

FIG. 3A is a bar graph showing luminescence in the supernatant of HEK293cells 24 hours after transfection with circRNA containing a panel ofviral 5′ UTR IRES sequences in GLuc (left bars, black) and FLuc (rightbars, gray) contexts (data presented as mean+SD, n=4).

FIG. 3B is a bar graph showing luminescence in the supernatant of HEK293cells 24 hours after transfection with circRNA containing a GLuc codingregion and a functional IRES. The effect of adding a polyA(30) orpolyAC(30) spacer sequence separating the IRES from the splice junctionis measured. (−): no spacer. pAC: 30 nt spacer consisting of adenosinesand cytosines. pA: 30 nt spacer consisting of adenosines (data presentedas mean+SD, n=4, *p<0.05).

FIG. 3C is a bar graph showing luminescence in the supernatant of HEK293(left, black) and HeLa (right, gray) cells 24 hours after transfectionwith the most effective circRNAs by IRES in b) (data presented asmean+SD, n=4).

FIG. 3D is a graph showing HPLC chromatogram of linear GLuc RNA (top)and a CVB3-GLuc-pAC splicing reaction (bottom).

FIG. 3E shows agarose gel of CVB3-GLuc-pAC purified by differentmethods. C: splicing reaction. +R: splicing reaction treated with RNaseR. +G,R: splicing reaction gel extracted, and then treated with RNase R.+H,R: splicing reaction HPLC purified, and then treated with RNase R.

FIG. 3F shows luminescence in the supernatant of HEK293 cells 24 hoursafter transfection with the CVB3-GLuc-pAC splicing reactions purified bydifferent methods as noted in FIG. 3E (data presented as mean+SD, n=4,*p<0.05).

FIG. 3G shows an agarose gel of HPLC fractions for the data in FIG. 3D.From left to right: Fraction 1, 2, 3, 4, 5.

FIG. 3H is a bar graph showing luminescence in the supernatant of HEK293(left) and HeLa (right) cells 24 hours after transfection withCVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA (datapresented as mean+SD, n=4 HEK293, n=3 HeLa, *p<0.05).

FIG. 3I is a graph showing luminescence in the supernatant of HEK293cells starting 24 hours after transfection with CVB3-GLuc-pAC circRNA ormodified or unmodified linear GLuc mRNA and continuing for 6 days (datapresented as mean+SD, n=4 HEK293).

FIG. 3J is a graph showing relative cumulative luminescence producedover 6 days by HEK293 (left) and HeLa (right) cells transfected withCVB3-GLuc-pAC circRNA or modified or unmodified linear GLuc mRNA (datapresented as mean+SD, n=4 HEK293, n=3 HeLa, *p<0.05).

FIG. 3K is a graph showing luminescence in the supernatant of HeLa cellsstarting 24 hours after transfection with CVB3-GLuc-pAC circRNA ormodified or unmodified linear GLuc mRNA and continuing for 6 days (datapresented as mean+SD, n=3 HeLa).

FIG. 4A shows the effect of insert length on RNA circularizationefficiency using a permuted group I intron containing optimized spacersand homology arms. 1.2: 1200 nt circRNA. 2.4: 2400 nt circRNA. 4.8: 4800nt circRNA.

FIG. 4B is a bar graph showing gel quantification of splicing efficiencyof precursor molecules containing different engineered sequences. WHA:weak homology arms. SHA: strong homology arms. DS: disruptive spacer.PS: permissive spacer. Ana: Anabaena base. IH: internal homology.

FIG. 5A is a bar graph showing gel quantification (ImageJ) of splicingefficiency and nicking in circRNA containing different interveningcoding regions, arranged by length. Splicing efficiency presented asratio of non-precursor (circular, nicked) to precursor RNA. Nickingpresented as ratio of nicked RNA to non-nicked long RNA (precursor,circular).

FIG. 5B shows agarose gel demonstrating the effect of small deletionsencompassing the 5′ and 3′ splice sites on splicing.

FIG. 5C is a bar graph showing luminescence in the supernatant of HEK293cells 24 hours after transfection with circRNA coding for GLuc andcontaining an EMCV IRES or the same precursor RNA with deleted splicesites (data presented as mean+SD, n=4).

FIG. 6A is a bar graph showing additional IRES sequences and putativeIRES sequences tested for functionality in the context of circRNA.

FIG. 6B is a schematic diagram of an RNAFold prediction of precursor RNAsecondary structure at the splice junction. IRES, coding region, andintrons are excluded.

FIG. 6C is a bar graph showing luminescence in the supernatant of Min6cells 24 hours after transfection with the most effective circRNAs byIRES in FIG. 3B (data presented as mean+SD, n=4).

FIG. 6D is a bar graph showing luminescence in the supernatant of A549cells 24 hours after transfection with the most effective circRNAs byIRES in FIG. 3B (data presented as mean+SD, n=4).

FIGS. 7A-7J. FIGS. 7A-F show an example of the design, synthesis, andpurification of circRNA. FIG. 7A) Precursor RNA design and self-splicingoverview. Colors denote different regions of the RNAs used. FIG. 7B)Schematics of RNAs introduced and used in this figure. ΔSplice Sites(ΔS) is identical to the precursor RNA except for small deletionsencompassing both splice sites. FIG. 7C) Agarose gel showing precursorRNA after splicing, RNase R digestion, HPLC purification, andoligonucleotide-guided RNase H digestion. Circular RNA is digested byRNase H into one major band, while ΔS is digested into two major bands,confirming circularity. FIG. 7D) Agarose gel showing cumulativepurification methods applied to circRNA. +RNase R: unpurified circRNAdigested with RNase R only. +HPLC: unpurified circRNA HPLC purified, andthen digested with RNase R. +Phos: unpurified circRNA HPLC purified,then treated with a phosphatase, and then digested with RNase R. FIG.7E) Cell viability, GLuc expression stability, and cytokine release from293 cells transfected with different circRNA preparations as describedin FIG. 7D). Cell viability was assessed 3 days after transfection.Cytokine release was assessed 24 hours after transfection (datapresented as mean+SD, n=3, ns=not significant p<0.05, ND=not detected).FIG. 7F) Cell viability, circRNA expression stability, and cytokinerelease from A549 cells transfected with different circRNA preparationsdescribed in FIG. 7D). Cell viability was assessed 3 days aftertransfection. Cytokine release was assessed 24 hours after transfection(data presented as mean+SD, n=3, ND=not detected, *p<0.05). FIGS. 7G-Jshow ΔSplice Sites (ΔS) characterization. FIG. 7G) Schematic of the RNAintroduced and used in this figure. ΔS is the linear circRNA precursorwith deleted splice sites (marked by x). ΔS is polyadenylated andtreated with phosphatase. FIG. 7H) Agarose gel showing ladder used toassign molecular weights and bands. ΔS does not detectably circularize.FIG. 7I) GLuc expression 24 hours after transfection of 293 cells withcircRNA or ΔS (data presented as mean+SD, n=3). FIG. 7J) GLuc proteinproduction stability over 3 days after transfection of 293 cells withcircRNA or ΔS (data presented as mean+SD, n=3).

FIGS. 8A-G. FIGS. 8A-F show splicing reaction fractionation andassessment of immunogenicity. FIG. 8A) Schematics of RNAs introduced andused in this figure. HMW: High Molecular Weight; this fraction containslinear and circular concatenations. FIG. 8B) Above: HPLC chromatogram ofan unpurified splicing reaction. Below: agarose gel of purifiedfractions. Adequate separation of precursor RNA was difficult, andtherefore ΔS was used instead. FIG. 8C) Cytokine release 24 hours aftertransfection of A549 cells with different HPLC fractions as described inFIG. 8B) (data presented as mean+SD, n=3, *p<0.05). FIG. 8D) Cellviability 36 hours after transfection of A549 cells with different HPLCfractions as described in b) (data presented as mean+SD, n=3, *p<0.05).FIG. 8E) RIG-I and IFN-β1 transcript induction 18 hours aftertransfection of A549 cells with the indicated RNAs. 3p-hpRNA is 5′triphosphate hairpin RNA and a specific agonist of RIG-I (data presentedas mean+SD, n=3, *p<0.05). FIG. 8F) RIG-I and IFN-β1 transcriptinduction 18 hours after transfection of A549 cells with RNase Rdigested splicing reactions or the late circRNA fraction (data presentedas mean+SD, n=3, *p<0.05). FIG. 8G shows additional cytokines assessedin culture media after transfection of A549 (left) and 293 (right) cellswith different circRNA preparations as described in FIG. 7D (datapresented as mean+SD, n=3). In most cases, these analytes were detectedat extremely low levels, precluding the observance of significantdifferences.

FIGS. 9A-O. FIGS. 9A-F show determination of circRNA immunogenicity inrelation to linear mRNA. FIG. 9A) Schematics of RNAs introduced and usedin this figure. Linear mRNAs do not contain an IRES or other structuredfeatures that may provoke a structure-specific immune response. FIG. 9B)Agarose gel showing progressive modification of circRNA precursor withm1ψ. FIG. 9C) Agarose gel showing purified unmodified and modified RNAs.Modification with m1ψ reduces apparent molecular weight. FIG. 9D) GLucexpression 24 hours after transfection of 293 or A549 cells withunmodified circRNA or m1ψ-circRNA (data presented as mean+SD, n=3). FIG.9E) Cell viability, GLuc expression stability, and cytokine release from293 cells transfected with unmodified or m1ψ linear mRNA or circRNA.Cell viability was assessed 3 days after transfection. Cytokine releasewas assessed 24 hours after transfection (data presented as mean+SD,n=3, ns=not significant p<0.05, ND=not detected). FIG. 9F) Cellviability, GLuc expression stability, and cytokine release from A549cells transfected with unmodified or m1ψ linear mRNA or circRNA. Cellviability was assessed 3 days after transfection. Cytokine release wasassessed 24 hours after transfection (data presented as mean+SD, n=3,ND=not detected, *p<0.05). FIG. 9G) depicts GLuc activity (RLU). FIG.9H) Additional cytokines assessed in culture media after transfection ofA549 cells with different HPLC fractions as shown in FIG. 9H (datapresented as mean+SD, n=3, *p<0.05). FIG. 9I Cell viability 36 hoursafter mock transfection or no transfection of 293 cells (left) andtranscript induction 24 hours after mock transfection or no transfectionof 293 cells (right; fold induction relative to untransfected; datapresented as mean+SD, n=2, ns=not significant). FIG. 9J) IL-6 and RANTESsecretion by A549 cells 24 hours after mock transfection withMessengerMax, or without transfection (data presented as mean+SD, n=3).FIG. 9K) RIG-I and IFN-β1 transcript induction 24 hours aftertransfection of A549 cells with purified circRNA containing a syntheticRIG-I ligand (3p-hpRNA) as a percentage of total RNA transfected (datapresented as mean+SD, n=3, *p<0.05). FIG. 9L) RIG-I and IFN-β1transcript induction 24 hours after transfection of HeLa cells with theindicated RNAs (data presented as mean+SD, n=3, *p<0.05). FIG. 9M) Timecourse of transcript induction 2-8 hours after transfection of 150,000A549 cells with 20 ng of the indicated RNAs (data presented as mean+SD,n=2). FIG. 9N) GLuc expression 24 hours after transfection of RAW264.7cells at 80% confluence with the indicated RNAs (left). GLuc expressionstability over 2 days (right; data presented as mean+SD, n=3).Transcript induction 24 hours after transfection of RAW264.7 cells withthe indicated RNAs (data presented as mean+SD, n=2). FIG. 9O) Analysisof a circular RNA containing an EMCV IRES and coding for GFP (circGFP).Agarose gel showing circGFP circularization and purified circGFP (left).A549 cell viability 36 hours after reverse transfection of 20,000 A549cells with 40 ng of circRNA (right). Transcript induction 24 hours afterreverse transfection of A549 cells with the indicated RNAs (bottom; datapresented as mean+SD, n=2, ns=not significant).

FIGS. 10A-I. FIGS. 10A-F show CircRNA evasion of TLRs. FIG. 10A)Schematics of RNAs introduced and used for TLR experiments. LinearizedcircRNAs contain all of the same sequence elements as spliced circRNAdue to deletions encompassing both the introns and homology arms. FIG.10B) SEAP expression 36 hours after transfection of TLR reporter cellswith the indicated RNAs relative to null controls (data presented asmean+SD, n=3, ns=not significant, *p<0.05). FIG. 10C) SEAP expression 36hours after transfection of TLR8 reporter cells with the late circRNAfraction relative to the null control. (−): media contains nonucleoside. C: media contains cytidine (3.5 mM). U: media containsuridine (3.5 mM); (data presented as mean+SD, n=3, ns=not significant,*p<0.05). FIG. 10D) Schematic of RNAs introduced and used for TLR nickedRNA experiments. FIG. 10E) Agarose gel showing alternative circRNAnicking strategies. FIG. 10F) SEAP expression 36 hours aftertransfection of TLR reporter cells with the indicated RNAs relative tonull controls (data presented as mean+SD, n=3, *p<0.05).

FIGS. 10G-I show splint ligation optimization. FIG. 10G) Splint ligationprecursor RNA design and splicing overview. FIG. 10H) Different splintsused for ligation. Of note, these optimizations were conducted with aplasmid containing an NaeI restriction cut site for linearization,leading to unwanted RNA side products (seen as extraneous bands inLigase(−) and Splint(−) conditions) forming during in vitrotranscription. This site was changed to XbaI for the GLuc and hEposplint ligations used in FIG. 9A-F and FIG. 10A-F. OH: overhang (5′,3′);Tm: melting temperature. FIG. 10I) shows optimization of circularizationconditions.

FIGS. 11A-F. FIGS. 11A-D show hEpo circRNA characterization in vivo.FIG. 11A) Serum hEpo expression 6 hours after injection of 350 ng ofunmodified or m1ψ linear mRNA or circRNA complexed with MessengerMaxinto visceral adipose tissue (data presented relative to molecularweight, mean+SD, n=3). FIG. 11B) Relative hEpo expression in serum over42 hours (data presented as mean+SD, n=3). FIG. 11C) Cytokines detectedin serum 6 hours after injection of 350 ng of the indicated RNAs intovisceral adipose (data presented as mean+SD, n=3, *p<0.05). FIG. 11D)Injection site demonstrated by injection of modified firefly luciferasemRNA complexed with MessengerMax. FIG. 11E shows Cell viability, GLucexpression stability, and cytokine release from 293 cells transfectedwith unmodified or m1ψ linear mRNA or circRNA. Cell viability wasassessed 3 days after transfection. Cytokine release was assessed 24hours after transfection (data presented as mean+SD, n=3, ns=notsignificant p<0.05, ND=not detected). FIG. 11F shows additionalcytokines assessed in culture media after transfection of 293 and A549cells with unmodified or m1ψ linear mRNA or circRNA (see FIG. 9E,9F;data presented as mean+SD, n=3). In most cases, these analytes weredetected at extremely low levels, precluding the observance ofsignificant differences.

FIGS. 12A-I. FIGS. 12A-F show LNP-circRNA characterization. FIG. 12A)Cryo-TEM image of LNP-circRNA. FIG. 12B) hEpo expression 24 hours aftertransfection of 293 cells with equimolar quantities of LNP-5moU-mRNA orunmodified LNP-circRNA (left) and hEpo protein expression stability over3 days (right; data presented as mean+SD, n=3). FIG. 12C) RIG-I andIFN-β1 transcript induction 24 hours after transfection of A549 cellswith LNP-5moU-mRNA or unmodified LNP-circRNA. TR: transfection reagentplus 200 ng RNA (MessengerMax); LNP(1×): 200 ng LNP-RNA; LNP(2×): 400 ngLNP-RNA; L: 5moU-mRNA; C: circRNA (data presented as mean+SD, n=3,ns=not significant, p<0.05). FIG. 12D) SEAP expression 48 hours aftertransfection of TLR reporter cells with the RNAs indicated in c),relative to null controls (data presented as mean+SD, n=3). FIG. 12E)Serum hEpo expression 6 hours after injection of 1.5 picomoles ofLNP-5moU-mRNA or unmodified LNP-circRNA into visceral adipose (datapresented as mean+SD, n=5 Linear 5moU, Circular; n=3 Mock). FIG. 12F)Relative hEpo expression in serum over 42 hours after injection withLNP-RNAs (data presented as mean+SD, n=5 Linear 5moU, Circular; n=3Mock). FIG. 12G) Agarose gel of the linear RNAs depicted in FIG. 9G.FIG. 12H) SEAP expression 36 hours after transfection of TLR8 reportercells with the tailed linear RNA shown in a) relative to null controlsin the presence or absence of varying concentrations of uridine (datapresented as mean+SD, n=2). FIG. 12I) Complete data from FIG. 10Fincluding an additional positive control.

FIG. 13 shows a graph of HPLC chromatogram of an unpurified hEposplicing reaction.

FIGS. 14A-D show hEpo circRNA characterization in vitro. FIG. 14A)Agarose gel showing purified unmodified and modified RNAs. FIG. 14B)hEpo expression 24 hours after transfection of 293 cells with equimolarquantities of m1ψ-mRNA or unmodified circRNA (data presented as mean+SD,n=3). FIG. 14C) Cell viability, hEpo protein production stability, and24 hour protein expression from 293 cells transfected with equal weightsof unmodified or m1ψ linear mRNA or circRNA. Cell viability was assessed36 hours after transfection (data presented as mean+SD, n=3). FIG. 14D)Cell viability, hEpo protein production stability, and 24 hour proteinexpression from A549 cells transfected with equal weights of unmodifiedor m1ψ linear mRNA or circRNA. Cell viability was assessed 36 hoursafter transfection (data presented as mean+SD, n=3).

FIG. 15 shows additional cytokines detected in serum 6 hours afterintraperitoneal injection of equal weights of the indicated RNAs (seeFIG. 10F; data presented as mean+SD, n=3). In most cases, these analyteswere detected at extremely low levels, precluding the observance ofsignificant differences.

FIGS. 16A-D show LNP-RNA characterization in vivo. FIG. 16A)Physicochemical properties of LNP-RNAs (data presented as mean±SD, n=3).FIG. 16B) Injection site demonstrated by injection of modified fireflyluciferase mRNA formulated into cKK-E12 LNPs. Luminescence detected at 6and 24 hours shows local delivery to visceral adipose. FIG. 16C)Cytokines detected in serum 6 hours after intraperitoneal injection of750 ng of the indicated RNAs formulated into cKK-E12 LNPs (datapresented as mean+SD, n=3). FIG. 16D) Transcript induction in visceraladipose tissue 24 hours after intraperitoneal injection of 750 ng of theindicated RNAs formulated into cKK-E12 LNPs (data presented as mean+SD,n=3). FIG. 16E) Serum hEpo expression from liver 6 hours afterintravenous injection of 0.1 mg/kg 5moU-mRNA or unmodified circRNA(left) and relative hEpo expression over 42 hours (right; data presentedrelative to molecular weight, mean+SD, n=3).

FIG. 17 shows circularization of precursor RNA containing a T4 phagepermuted intron, EMCV IRES, GLuc reading frame, and strong homology armsdirectly after in vitro transcription. Precursor RNA was heated at theindicated temperatures, cooled on ice, and then spliced at 55 degreesCelsius.

FIGS. 18A-18C show circularization of precursor RNA. FIG. 18A showscircularization of precursor RNA containing a T4 or Anabaena permutedintron, EMCV IRES, GLuc reading frame, strong homology arms, and a 5′spacer at different GTP concentrations.

FIG. 18B shows circularization of the precursor RNAs described in FIG.18A at different concentrations of RNA. FIG. 18C shows gel extraction ofmajor top and bottom bands resulting from complete splicing using threealternative protocols to rule out interconversion of species.

FIG. 19 shows a graph of stability and expression of GLuc fromEMCV-circRNA without spacers or linear mRNA over 144 h in 293 cells.

FIGS. 20A-20D. FIG. 20A) Stability and expression of GLuc fromEMCV-circRNA without spacers and with or without UTRs over 144 h in HeLacells. FIG. 20B) Stability and expression of GLuc from EMCV-circRNAwithout spacers and with or without UTRs over 144 h in 293 cells. FIG.20C) Expression of GLuc from CVB3-circRNA with a 5′ spacer and with orwithout different UTRs. Trilink: 5mC/pseudo-modified linear mRNApurchased from Trilink. FIG. 20D) Circularization of precursor RNAcontaining a T4 permuted intron, EMCV IRES, GLuc reading frame, stronghomology arms, a 5′ spacer with or without different UTRs. R: RNase Rdigestion.

FIGS. 21A-21G. FIGS. 21A-21D) Expression of GLuc from circRNA with a 5′spacer and with different IRES sequences in 293 and HeLa cells. 21E-21F)Expression of GLuc from circRNA with a 5′ spacer and with different IRESsequences or UTRs in 293 and HeLa cells. CircRNAs contain a CVB3 IRESunless otherwise stated. Trilink: 5mC/pseudo-modified linear mRNApurchased from Trilink. 21G) Comparison of StemFect transfection reagentand lipid nanoparticle (LNP) delivery of different RNA species in 293cells.

FIGS. 22A-22B. FIGS. 22A-22B) Expression of GLuc from CVB3-circRNA withan Anabaena permuted intron, a 5′ spacer, and with or without differentpolyN sequences in 293 and HeLa cells.

FIGS. 23A-23B. FIG. 23A) Comparison of the effects of permuted intronsequence context on the expression of GLuc from circRNA with a 5′ spacerand the indicated IRES in 293 cells. FIG. 23B) Serum expression of GLucfrom circRNA containing a T4 permuted intron, a 5′ spacer, and differentIRES sequences, or linear mRNA. RNA was formulated into liver-homingLNPs and injected intravenously. Serum was collected 6 hours afterinjection.

FIG. 24 shows expression of GLuc from a plasmid containing circRNA with5′ and 3′ spacers and a CVB3 IRES in HeLa cells. Base plasmid does notcontain the CVB3 IRES. dSplice Sites contains mutated splice sites toabrogate circularization after transcription.

FIGS. 25A-25B. FIG. 25A) Circularization of precursor RNA containing anAnabaena permuted intron, GLuc reading frame, strong homology arms, 5′and 3′ spacers, and the indicated IRES. FIG. 25B) Circularization ofprecursor RNA containing an Anabaena permuted intron, FLuc readingframe, strong homology arms, 5′ and 3′ spacers, and the indicated IRES.

FIGS. 26A-26B. FIG. 26A) HeLa cells. FIG. 26B) A594 cells. RIG-I andIFNB1 fold induction after transfection of indicated circRNApreparations. All preparations contain circRNA with an Anabaena permutedintron, GLuc reading frame, strong homology arms, 5′ and 3′ spacers, anda CVB3 IRES. Unpurified: total splicing reaction. GMP: CircRNAprecursors transcribed in the presence of 12.5-fold GMP over GTP.3phpRNA: triphosphate hairpin RNA positive control.

DETAILED DESCRIPTION

A description of example embodiments follows.

As described herein, exogenous circRNA was developed to extend theduration of protein expression from full-length RNA messages. First, aself-splicing intron was engineered to circularize efficiently a widerange of RNAs in vitro, coding for proteins such as Cas9, by rationallydesigning ubiquitous accessory sequences that aid in splicing.Functional protein was produced from these circRNAs in eukaryotic cellsand translation incorporating different internal ribosome entry sites(IRES) and internal polyadenosine tracts was maximized. EngineeredcircRNA purified by high performance liquid chromatography displayedexceptional protein production qualities in terms of both quantity ofprotein produced and stability of production. Provided herein aremethods and compositions that facilitate the use of exogenous circRNAfor robust and stable protein expression in eukaryotic cells, renderingcircRNA a promising alternative to linear mRNA.

Circular RNAs (circRNAs) endogenous to eukaryotic cells have drawnincreasing interest due to their prevalence and range of potentialbiological functions (Barrett, S. P. & Salzman, J., “Circular RNAs:analysis, expression and potential functions,” Development,143(11):1838-1847 (2016)). Most circRNAs are generated throughbacksplicing and appear to fulfill noncoding roles (Barrett, S. P. &Salzman, J., “Circular RNAs: analysis, expression and potentialfunctions,” Development, 143(11):1838-1847 (2016); Chen, L. & Yang, L.,“Regulation of circRNA biogenesis,” RNA Biology, 12(4):381-388 (2015);Jeck, W. R. and Sharpless, N. E., “Detecting and characterizing circularRNAs,” Nat. Biotechnol., 32:453-461 (2014); Wang, Y. & Wang, Z.,“Efficient backsplicing produces translatable circular mRNAs,” RNA,21(2):172-179 (2014); Hansen, T. B. et al., “Natural RNA circlesfunction as efficient microRNA sponges,” Nature, 495(7441):384-388(2013); Li, Z. et al., “Exon-intron circular RNAs regulate transcriptionin the nucleus,” Nature Structural & Molecular Biology, 22(3):256-264(2015)). However, it has been suggested that some circRNAs endogenous toDrosophila may be translated into protein (Legnini, I. et al.,“Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions inMyogenesis,” Molecular Cell, 66(1):22-37.e9 (2017); Pamudurti, N. R. etal., “Translation of CircRNAs,” Molecular Cell, 66(1) (2017)).

In addition to having protein-coding potential, endogenous circRNAs lackthe free ends necessary for exonuclease-mediated degradation, renderingthem resistant to several mechanisms of RNA turnover and granting themextended lifespans as compared to their linear mRNA counterparts (Chen,L. & Yang, L., “Regulation of circRNA biogenesis,” RNA Biology,12(4):381-388 (2015); Enuka, Y. et al., “Circular RNAs are long-livedand display only minimal early alterations in response to a growthfactor,” Nucleic Acids Research, 44(3):1370-1383 (2015)). For thisreason, circularization may allow for the stabilization of mRNAs thatgenerally suffer from short half lives and may therefore improve theoverall efficacy of exogenous mRNA in a variety of applications(Kaczmarek, J. C. et al., “Advances in the delivery of RNA therapeutics:from concept to clinical reality,” Genome Medicine, 9(1) (2017); Fink,M. et al., “Improved translation efficiency of injected mRNA duringearly embryonic development,” Developmental Dynamics, 235(12):3370-3378(2006); Ferizi, M., et al., “Stability analysis of chemically modifiedmRNA using micropattern-based single-cell arrays,” Lab Chip,15(17):3561-3571 (2015)). However, the efficient circularization of longin vitro transcribed (IVT) RNA, the purification of circRNA, and theadequate expression of protein from circRNA are significant obstaclesthat must be overcome before their protein-coding potential can berealized. As described herein, in one embodiment, an engineeringapproach is presented to generate exogenous circRNAs for potent anddurable protein expression in cells, e.g., eukaryotic cells.

Abbreviations

GFP Green fluorescent protein

ORF Open reading frame

IRES Internal ribosome entry site

UTR Untranslated region

HEK Human embryonic kidney

IRES Internal Ribosome Entry Site

EMCV Encephalomyocarditis virus, a picornavirus

PIE permutated intron-exon splice site

In one embodiment, the present invention is directed to a vector formaking circular RNA, said vector comprising the following elementsoperably connected to each other and arranged in the following sequence:a.) a 5′ homology arm, b.) a 3′ group I intron fragment containing a 3′splice site dinucleotide, c.) optionally, a 5′ spacer sequence, d.)optionally, an internal ribosome entry site (IRES), e.) a protein codingor noncoding region, f.) optionally, a 3′ spacer sequence, g.) a 5′group I intron fragment containing a 5′ splice site dinucleotide, andh.) a 3′ homology arm, said vector allowing production of a circular RNAthat is translatable and/or biologically active inside eukaryotic cells.

As used herein, the lettering of the elements (e.g., “a.)-h.)”) are usedsolely for clarity purposes. In addition, it is understood that inalternative embodiments, it is possible that the elements can bearranged in a different sequence, and/or that one or more elements maybe omitted.

As used herein, the elements of a vector are “operably connected” ifthey are positioned on the vector such that they can be transcribed toform a precursor RNA that can then be circularized into a circular RNAusing the methods provided herein.

In one embodiment, the present invention is directed to a vector (e.g.,a plasmid) for making circRNA, said vector comprising the followingelements operably connected to each other and arranged in the followingsequence: a.) a 5′ homology arm, b.) a 3′ group I intron fragment, c.)an optional 5′ spacer sequence, d.) an optional internal ribosome entrysite (IRES), e.) a protein coding or noncoding region, f.) an optional3′ spacer sequence, g.) a 5′ group I intron fragment containing a 5′splice site dinucleotide, and h.) a 3′ homology arm, said vectorallowing production of a circRNA that is translatable or biologicallyactive inside eukaryotic cells.

As used herein, a “homology arm” is any contiguous sequence that is 1)predicted to form base pairs with at least about 75% (e.g., at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,about 100%) of another sequence in the RNA, such as another homology arm2) at least 7 nt long and no longer than 250 nt 3) located before andadjacent to, or included within, the 3′ intron fragment and/or after andadjacent to, or included within, the 5′ intron fragment and, optionally,4) predicted to have less than 50% (e.g., less than 45%, less than 40%,less than 35%, less than 30%, less than 25%) base pairing withunintended sequences in the RNA (e.g., non-homology arm sequences). A“strong homology arm” refers to a homology arm with a Tm of greater than50 degrees Celsius when base paired with another homology arm in theRNA.

As used herein, a 3′ group I intron fragment is a contiguous sequencethat is at least 75% (e.g., at least 80%, at least 85%, at least 90%, atleast 95%, 100%) homologous to a 3′ proximal fragment of a natural groupI intron, including the 3′ splice site dinucleotide, and, optionally,the adjacent exon sequence at least 1 nucleotide in length (e.g., atleast 5 nucleotides in length, at least 10 nucleotides in length, atleast 15 nucleotides in length, at least 20 nucleotides in length, atleast 25 nucleotides in length, at least 50 nucleotides in length). Inone embodiment, the included adjacent exon sequence is about the lengthof the natural exon. In some embodiments, a 5′ group I intron fragmentis a contiguous sequence that is at least 75% (e.g., at least 80%, atleast 85%, at least 90%, at least 95%, 100%) homologous to a 5′ proximalfragment of a natural group I intron, including the 5′ splice sitedinucleotide and, optionally, the adjacent exon sequence at least 1nucleotide in length (e.g., at least 5 nucleotides in length, at least10 nucleotides in length, at least 15 nucleotides in length, at least 20nucleotides in length, at least 25 nucleotides in length, at least 50nucleotides in length). In one embodiment, the included adjacent exonsequence is about the length of the natural exon.

As used herein, a “spacer” refers to any contiguous nucleotide sequencethat is 1) predicted to avoid interfering with proximal structures, forexample, from the IRES, coding or noncoding region, or intron 2) atleast 7 nucleotides long (and optionally no longer than 100 nucleotides)3) located downstream of and adjacent to the 3′ intron fragment and/orupstream of and adjacent to the 5′ intron fragment and/or 4) containsone or more of the following: a) an unstructured region at least 5 ntlong b) a region predicted base pairing at least 5 nt long to a distal(i.e., non-adjacent) sequence, including another spacer, and/or c) astructured region at least 7 nt long limited in scope to the sequence ofthe spacer.

As used herein, “interfering” with regard to sequences refers tosequence(s) predicted or empirically determined to alter the folding ofother structures in the RNA, such as the IRES or group I intron-derivedsequences.

As used herein, “unstructured” with regard to RNA refers to an RNAsequence that is not predicted by the RNAFold software or similarpredictive tools to form a structure (e.g., a hairpin loop) with itselfor other sequences in the same RNA molecule.

As used herein, “structured” with regard to RNA refers to an RNAsequence that is predicted by the RNAFold software or similar predictivetools to form a structure (e.g., a hairpin loop) with itself or othersequences in the same RNA molecule.

In some embodiments, the spacer sequence can be, for example, at least10 nucleotides in length, at least 15 nucleotides in length, or at least30 nucleotides in length. In some embodiments, the spacer sequence is atleast 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30nucleotides in length. In some embodiments, the spacer sequence is nomore than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides inlength. In some embodiments the spacer sequence is between 20 and 50nucleotides in length. In certain embodiments, the spacer sequence is10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides in length.

The spacer sequences can be polyA sequences, polyA-C sequences, polyCsequences, or poly-U sequences, or the spacer sequences can bespecifically engineered depending on the IRES. Spacer sequences asdescribed herein can have two functions: (1) promote circularization and(2) promote functionality by allowing the introns and IRES to foldcorrectly. More specifically, the spacer sequences as described hereinwere engineered with three priorities: 1) to be inert with regards tothe folding of proximal intron and IRES structures; 2) to sufficientlyseparate intron and IRES secondary structures; and 3) to contain aregion of spacer-spacer complementarity to promote the formation of a‘splicing bubble’. In one embodiment, the vectors are compatible withmany possible IRES and coding or noncoding regions and two spacersequences.

In some embodiments, an RNA folding computer software, such as RNAFold,can be utilized to guide designs of the various elements of the vector,including the spacers.

In some embodiments, one or more elements in the vector for makingcircular RNA comprise at least 75% sequence identity with naturalsequences, including e.g., the IRES and intron fragment elements. Insome embodiments, the protein coding regions or noncoding regions arenot naturally occurring nucleotide sequences. In some embodiments, theprotein coding regions encode natural or synthetic proteins.

In some embodiments, the coding or noncoding regions can be natural orsynthetic sequences. In some embodiments, the coding regions can encodechimeric antigen receptors, immunomodulatory proteins, and/ortranscription factors. In some embodiments, the noncoding regions canencode sequences can alter cellular behavior, such as e.g., lymphocytebehavior. In some embodiments, the noncoding sequences are antisense tocellular RNA sequences.

In one embodiment, the vector can comprise a 5′ spacer sequence, but nota 3′ spacer sequence. In another embodiment, the vector can comprise a3′ spacer sequence, but not a 5′ spacer sequence. In another embodiment,the vector can comprise neither a 5′ spacer sequence, nor a 3′ spacersequence. In another embodiment, the vector does not comprise an IRESsequence. In a further embodiment, the vector does not comprise an IRESsequence, a 5′ spacer sequence or a 3′ spacer sequence.

As used herein, a “vector” means a piece of DNA, that is synthesized(e.g., using PCR), or that is taken from a virus, plasmid, or cell of ahigher organism into which a foreign DNA fragment can be or has beeninserted for cloning and/or expression purposes. In some embodiments, avector can be stably maintained in an organism. A vector can comprise,for example, an origin of replication, a selectable marker or reportergene, such as antibiotic resistance or GFP, and/or a multiple cloningsite (MCS). The term includes linear DNA fragments (e.g., PCR products,linearized plasmid fragments), plasmid vectors, viral vectors, cosmids,bacterial artificial chromosomes (BACs), yeast artificial chromosomes(YACs), and the like. In one embodiment, the vectors provided hereincomprise a multiple cloning site (MCS). In another embodiment, thevectors provided herein do not comprise a MCS.

Examples of Group I intron self-splicing sequences include, but are notlimited to, self-splicing permuted intron-exon sequences derived from T4bacteriophage gene td or Cyanobacterium Anabaena sp. pre-tRNA-Leu gene.

The protein coding region can encode a protein of eukaryotic orprokaryotic origin. In some embodiments, the protein can be any proteinfor therapeutic use or diagnostic use. For example, the protein codingregion can encode human protein or antibodies. In some embodiments, theprotein can be selected from, but not limited to, hFIX, SP-B, VEGF-A,human methylmalonyl-CoA mutase (hMUT), CFTR, cancer self-antigens, andadditional gene editing enzymes like Cpfl, zinc finger nucleases (ZFNs)and transcription activator-like effector nucleases (TALENs). In someembodiments, the vector or circRNA lacks a protein coding sequence. Insome embodiments, the precursor RNA is a necessary intermediate betweenplasmid and circRNA.

The 5′ and 3′ homology arms can be synthetic sequences and are distinctfrom the internal homology regions but similar in function. The homologyarms can be, e.g., about 5-50 nucleotides in length, about 9-19nucleotides in length, for example, about 5, about 10 about 20, about30, about 40, or about 50 nucleotides in length. In another embodiment,the homology arms can be 9 nucleotides in length. In a furtherembodiment, the homology arms can be 19 nucleotides in length. In someembodiments, the homology arms are at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments,the homology arms are no more than 50, 45, 40, 35, 30, 25 or 20nucleotides in length. In some embodiments, the homology arms are 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 or 50 nucleotides in length.

In some embodiments, the vector comprises an IRES sequence. The IRESsequence can be selected from, but not limited to, an IRES sequence of aTaura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus,simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus,Reticuloendotheliosis virus, fuman poliovirus 1, Plautia stali intestinevirus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulatavirus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulatavirus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus,Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71,Equine rhinitis virus, Ectropis obliqua picorna-like virus,Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, BlackQueen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitisvirus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus,Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1,Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, HumanBCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L,Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1,Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophilareaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, HumanVEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S.cerevisiae YAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnipcrinkle virus, an aptamer to eIF4G, Coxsackievirus B3 (CVB3) orCoxsackievirus A (CVB1/2). Wild-type IRES sequences can also be modifiedand be effective in the invention. In some embodiments, the IRESsequence is about 50 nucleotides in length.

In some embodiments, in order to express protein in a cell, the circularRNA can be transfected into the cell using, for example, lipofection orelectroporation. In another embodiment, the circular RNA is transfectedinto a cell using a nanocarrier. The nanocarrier can be, for example, alipid, polymer or a lipo-polymeric hybrid.

The circular RNA can be purified by the method of running the RNAthrough a size-exclusion column in tris-EDTA or citrate buffer in ahigh-performance liquid chromatography (HPLC) system. In one embodiment,the RNA is run through the size-exclusion column in tris-EDTA or citratebuffer at pH in the range of about 4-7 at a flow rate of about 0.01-5mL/minute.

In certain embodiments, provided herein is a method of generatingprecursor RNA by performing in vitro transcription using a vectorprovided herein as a template (e.g., a vector provided herein with a RNApolymerase promoter positioned upstream of the 5′ homology arm).

In some embodiments, the use of a nucleotide, nucleoside, or achemically modified nucleotide or nucleoside in the in vitrotranscription reactions described herein is at an excess concentrationrelative to the analogous nucleotide triphosphate. “Excessconcentration” is defined as greater than the concentration of theanalogous nucleotide triphosphate, with the purpose of changing the 5′end nucleotide, specifically to reduce the immunogenicity of circRNApreparations by preventing the inclusion of a 5′ triphosphate motif orto allow for the enzymatic circularization of precursor molecules byincluding the necessary 5′ monophosphate motif.

In some embodiments, the nucleotide used in excess is guanosinemonophosphate (GMP). In other embodiments, the nucleotide used in excessis GDP, ADP, CDP, UDP, AMP, CMP, UMP, guanosine, adenosine, cytidine,uridine, or any chemically modified nucleotide or nucleoside. In someembodiments, the excess is about a 10-fold excess. In some embodiments,the excess is about a 12.5-fold excess.

In one embodiment, the nucleotide, nucleoside, or a chemically modifiednucleotide or nucleoside is used at concentrations at least about 10× inexcess of the analogous nucleotide triphosphate in the in vitrotranscription reaction.

In some embodiments, the circRNA that results from precursor RNAsynthesized in the presence of a nucleotide, nucleoside, or a chemicallymodified nucleotide or nucleoside at least about 10× in excess of theanalogous nucleotide triphosphate in the in vitro transcription reactionis then purified by HPLC to achieve minimal immunogenicity.

Pharmaceutical Compositions/Administration

In embodiments of the present disclosure, the circRNA products describedherein and/or produced using the vectors and/or methods describedherein, may be provided in compositions, e.g., pharmaceuticalcompositions.

Therefore, in some embodiments, the invention also relates tocompositions, e.g., compositions comprising a circRNA (circRNA product)and a pharmaceutically acceptable carrier. In one aspect, the presentdisclosure provides pharmaceutical compositions comprising an effectiveamount of a circRNA described herein and a pharmaceutically acceptableexcipient. Pharmaceutical compositions of the present disclosure maycomprise a circRNA as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, excipients ordiluents. In some embodiments, pharmaceutical compositions of thepresent disclosure may comprise a circRNA expressing cell, e.g., aplurality of circRNA-expressing cells, as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, excipients or diluents.

In some embodiments, a pharmaceutically acceptable carrier can be aningredient in a pharmaceutical composition, other than an activeingredient, which is nontoxic to the subject.

A pharmaceutically acceptable carrier can include, but is not limitedto, a buffer, excipient, stabilizer, or preservative. Examples ofpharmaceutically acceptable carriers are solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible, suchas salts, buffers, saccharides, antioxidants, aqueous or non-aqueouscarriers, preservatives, wetting agents, surfactants or emulsifyingagents, or combinations thereof. The amounts of pharmaceuticallyacceptable carrier(s) in the pharmaceutical compositions may bedetermined experimentally based on the activities of the carrier(s) andthe desired characteristics of the formulation, such as stability and/orminimal oxidation.

In some embodiments, such compositions may comprise buffers such asacetic acid, citric acid, histidine, boric acid, formic acid, succinicacid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Trisbuffers, HEPPSO, HEPES, neutral buffered saline, phosphate bufferedsaline and the like; carbohydrates such as glucose, sucrose, mannose, ordextrans, mannitol; proteins; polypeptides or amino acids such asglycine; antioxidants; chelating agents such as EDTA or glutathione;adjuvants (e.g., aluminum hydroxide); antibacterial and antifungalagents; and preservatives.

In certain embodiments, compositions of the present disclosure can beformulated for a variety of means of parenteral or non-parenteraladministration. In one embodiment, the compositions can be formulatedfor infusion or intravenous administration. Compositions disclosedherein can be provided, for example, as sterile liquid preparations,e.g., isotonic aqueous solutions, emulsions, suspensions, dispersions,or viscous compositions, which may be buffered to a desirable pH.Formulations suitable for oral administration can include liquidsolutions, capsules, sachets, tablets, lozenges, and troches, powdersliquid suspensions in an appropriate liquid and emulsions.

In one aspect, the disclosure relates to administering a therapeuticallyeffective amount of a composition comprising a circRNA described hereinfor the treatment of a subject having, or at risk of developing, adisease or disorder, e.g., cancer. In another aspect, the disclosurerelates to administering a therapeutically effective amount of acomposition comprising a circRNA described herein for the treatment of asubject having a disease involving loss of a functional gene.

In some embodiments, the treatment aims to prolong translation from thecircRNA to a protein.

Pharmaceutical compositions of the present disclosure may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the subject, and the typeand severity of the subject's disease, although appropriate dosages maybe determined by clinical trials.

The terms “treat” or “treatment” refer to therapeutic treatment whereinthe object is to slow down (lessen) an undesired physiological change ordisease, or provide a beneficial or desired clinical outcome duringtreatment. Beneficial or desired clinical outcomes include alleviationof symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and/or remission(whether partial or total), whether detectable or undetectable.“Treatment” can also mean prolonging survival as compared to expectedsurvival if a subject was not receiving treatment. Those in need oftreatment include those subjects already with the undesiredphysiological change or disease as well as those subjects prone to havethe physiological change or disease.

A “therapeutically effective amount” or “effective amount”, usedinterchangeably herein, refers to an amount effective, at dosages andfor periods of time necessary, to achieve a desired therapeutic result.A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of a therapeutic or a combination of therapeutics to elicit adesired response in the individual. Example indicators of an effectivetherapeutic or combination of therapeutics that include, for example,improved well-being of the patient, reduction of disease burden,arrested or slowed progression of disease, and/or absence of progressionof disease to other locations in the body.

As used herein, the term “subject” refers to an animal. The terms“subject” and “patient” may be used interchangeably herein. As such, a“subject” includes a human that is being treated for a disease, orprevention of a disease, such as a patient.

As used herein, the term “splice site dinucleotide” refers to the twonucleotides that border a splice site.

In some embodiments, the method described herein may be used to treat ananimal subject belonging to any classification. Examples of such animalsinclude mammals, such as mice, hamsters, rabbits. cats, dogs, cows, pigsor horses). The mammals may be of monkeys, humans and apes. In oneembodiment, the mammal is a human.

Delivery systems useful in the context of embodiments of the inventionmay include time-released, delayed release, and sustained releasedelivery systems such that the delivery of the compositions occurs priorto, and with sufficient time to cause, sensitization of the site to betreated. The composition can be used in conjunction with othertherapeutic agents or therapies. Such systems can avoid repeatedadministrations of the composition, thereby increasing convenience tothe subject and the physician, and may be particularly suitable forcertain composition embodiments of the invention.

Release delivery systems include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polyesteramides,polyorthoesters, polycaprolactones, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-di- and tri-glycerides; sylastic systems; peptide based systems;hydrogel release systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. In some embodiments, lipid nanoparticles or polymers are used asdelivery vehicles for therapeutic circRNAs described herein, includingdelivery of RNA to tissues.

In certain embodiments, the administration of the compositions may becarried out in any manner, e.g., by parenteral or nonparenteraladministration, including by aerosol inhalation, injection, infusions,ingestion, transfusion, implantation or transplantation. For example,the compositions described herein may be administered to a patienttrans-arterially, intradermally, subcutaneously, intratumorally,intramedullary, intranodally, intramuscularly, by intravenous (i.v.)injection, intranasally, intrathecally or intraperitoneally. In oneaspect, the compositions of the present disclosure are administeredintravenously. In one aspect, the compositions of the present disclosureare administered to a subject by intradermal or subcutaneous injection.The compositions may be injected, for instance, directly into a tumor,lymph node, tissue, organ, or site of infection.

In one embodiment, administration may be repeated after one day, twodays, three days, four days, five days, six days, one week, two weeks,three weeks, one month, five weeks, six weeks, seven weeks, two months,three months, four months, five months, six months or longer. Repeatedcourses of treatment are also possible, as is chronic administration.The repeated administration may be at the same dose or at a differentdose.

In some embodiments, the compositions may be administered in the methodsof the invention by maintenance therapy, such as, e.g., once a week fora period of 6 months or more.

In one embodiment, cells can transiently express the circRNA describedherein for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days afterintroduction. Transient expression of the circRNA can be affected by themethod of delivery. In one embodiment, the circRNA is transduced intothe cell by electroporation. In one embodiment, the circRNA isintroduced into the cell by lipid transfection methods known in the art.

In some embodiments, a circRNA as described herein may be used incombination with other known agents and therapies. Administered “incombination”, as used herein, means that two (or more) differenttreatments are delivered to the subject during the course of thesubject's treatment e.g., the two or more treatments are delivered afterthe subject has been diagnosed with the disease and before the diseasehas been cured or eliminated or treatment has ceased for other reasons.In some embodiments, the delivery of one treatment is still occurringwhen the delivery of the second begins, so that there is overlap interms of administration. This is sometimes referred to herein as“simultaneous” or “concurrent delivery”. In other embodiments, thedelivery of one treatment ends before the delivery of the othertreatment begins. In some embodiments of either case, the treatment ismore effective because of combined administration. For example, thesecond treatment is more effective, e.g., an equivalent effect is seenwith less of the second treatment, or the second treatment reducessymptoms to a greater extent, than would be seen if the second treatmentwere administered in the absence of the first treatment, or theanalogous situation is seen with the first treatment. In someembodiments, delivery is such that the reduction in a symptom, or otherparameter related to the disorder is greater than what would be observedwith one treatment delivered in the absence of the other. The effect ofthe two treatments can be partially additive, wholly additive, orgreater than additive. The delivery can be such that an effect of thefirst treatment delivered is still detectable when the second isdelivered.

In further embodiments, a composition described herein may be used in atreatment regimen in combination with surgery, radiation, chemotherapy,antibodies, or other agents.

EXAMPLES Example 1

There are three general strategies for exogenous RNA circularization:chemical methods using cyanogen bromide or a similar condensing agent,enzymatic methods using RNA or DNA ligases, and ribozymatic methodsusing self-splicing introns (Petkovic, S. & Muller, S., “RNAcircularization strategies in vivo and in vitro,” Nucleic AcidsResearch, 43(4):2454-2465 (2015); Beadudry, D. & Perreault, J., “Anefficient strategy for the synthesis of circular RNA molecules,” NucleicAcids Research, 23(15):3064-3066 (1995); Micura, R., “CyclicOligoribonucleotides (RNA) by Solid-Phase Synthesis,” Chemistry—AEuropean Journal, 5(7):2077-2082 (1999)). A ribozymatic method utilizinga permuted group I catalytic intron has been reported to be moreapplicable to long RNA circularization and requires only the addition ofGTP and Mg2+ as cofactors (Petkovic, S. & Muller, S., “RNAcircularization strategies in vivo and in vitro,” Nucleic AcidsResearch, 43(4):2454-2465 (2015)). This permuted intron-exon (PIE)splicing strategy consists of fused partial exons flanked by half-intronsequences (Puttaraju, M. & Been, M., “Group I permuted intron-exon (PIE)sequences self-splice to produce circular exons,” Nucleic AcidsResearch, 20(20):5357-5364 (1992)). In vitro, these constructs undergothe double transesterification reactions characteristic of group Icatalytic introns, but because the exons are already fused they areexcised as covalently 5′ to 3′ linked circles (FIG. 1A) (Petkovic, S. &Muller, S., “RNA circularization strategies in vivo and in vitro,”Nucleic Acids Research, 43(4):2454-2465 (2015)). Using this strategy asa starting point for creating a protein coding circular RNA, a 1.1 kbsequence containing a full-length encephalomyocarditis virus (EMCV)IRES, a Gaussia luciferase (GLuc) message, and two short regionscorresponding to exon fragments (E1 and E2) of the PIE construct betweenthe 3′ and 5′ introns of the permuted group I catalytic intron in thethymidylate synthase (Td) gene of the T4 phage were inserted (FIG. 1A,Table 1) (Ford, E. & Ares, M., “Synthesis of circular RNA in bacteriaand yeast using RNA cyclase ribozymes derived from a group I intron ofphage T4,” Proceedings of the National Academy of Sciences,91(8):3117-3121 (1994)). Precursor RNA was synthesized by run-offtranscription and then heated in the presence of magnesium ions and GTPto promote circularization, essentially as described previously for thecircularization of shorter RNAs (Ford, E. & Ares, M., “Synthesis ofcircular RNA in bacteria and yeast using RNA cyclase ribozymes derivedfrom a group I intron of phage T4,” Proceedings of the National Academyof Sciences, 91(8):3117-3121 (1994)). However, splicing products werenot obtained. It was speculated that long intervening regions betweensplice sites may reduce the ability of the splice sites to interact withone another and form a stable complex, thus reducing splicingefficiency. Indeed, the intervening region between the 5′ and 3′ splicesites of native group I introns is on average 300-500 nucleotides long,while the intervening region of the engineered RNA that we constructedwas two to four-fold longer (Vicens, Q., et al., “Toward predictingself-splicing and protein-facilitated splicing of group I introns,” RNA,14(10):2013-2029 (2008)). Therefore, perfectly complementary ‘homologyarms’ 9 (weak) or 19 (strong) nucleotides in length were designed andplaced at the 5′ and 3′ ends of the precursor RNA with the aim ofbringing the 5′ and 3′ splice sites into proximity of one another (FIG.1B, Table 1). Addition of these homology arms increased splicingefficiency from 0% to 16% for weak homology arms and to 48% for stronghomology arms as assessed by disappearance of the precursor RNA band(FIG. 1C). To ensure that the major splicing product was circular, thesplicing reaction was treated with RNase R (FIG. 1D). Sequencing acrossthe putative splice junction of RNase R-treated splicing reactionsrevealed ligated exons, and digestion of the RNase R-treated splicingreaction with oligonucleotide-targeted RNase H produced a single band incontrast to two bands yielded by RNase H-digested linear precursor (FIG.1D and FIG. 1E). These data show that circRNA is a major product ofthese splicing reactions and that agarose gel electrophoresis allows forsimple and effective separation of circular splicing products fromlinear precursor molecules, nicked circles, splicing intermediates, andexcised introns.

In order to further improve the efficiency of circRNA generation fromthe self-splicing precursor RNA, other factors that may influencesuccessful circularization were considered. The 3′ PIE splice site isproximal to the IRES, and because both sequences are highly structuredit was hypothesized that sequences within the IRES may interfere withthe folding of the splicing ribozyme, either proximally at the 3′ splicesite or distally at the 5′ splice site through long-distance contacts.In order to allow these structures to fold independently, a series ofspacers between the 3′ PIE splice site and the IRES were designed and itwas predicted would either permit or disrupt splicing (FIG. 1F, Table1). Permissive spacers were designed to conserve secondary structurespresent within intron sequences that may be important for ribozymeactivity, while the disruptive spacer was designed to disrupt sequencesin both intron halves, especially the 5′ half. The addition of spacersequences predicted to permit splicing increased splicing efficiencyfrom 46% to 87% (P1 and P2), while the addition of a disruptive spacersequence completely abrogated splicing (FIG. 1G). This improvedconstruct, containing both homology arms and rationally designedspacers, was able to circularize RNA approaching 5 kb in length (FIG.4). The use of an alternative group I catalytic intron from the Anabaenapre-tRNA was also explored (Puttaraju, M. & Been, M., “Group I permutedintron-exon (PIE) sequences self-splice to produce circular exons,”Nucleic Acids Research, 20(20):5357-5364 (1992)). The same optimizationtechniques used to increase the efficiency of the permuted T4 phageintron splicing reaction were applied. Interestingly, during ouroptimizations it was noted that switching from the T4 catalytic intronto the Anabaena catalytic intron may have resulted in the weakening of ashort stretch of internal homology between the IRES and the 3′ end ofthe coding region, which may have aided in the formation of an isolated‘splicing bubble’ (FIG. 1H). Strengthening this internal homologyfurther increased splicing efficiency from 84% to 95% using the permutedAnabaena catalytic intron (FIG. 1H and FIG. 1I, Table 1). The use of theAnabaena catalytic intron resulted in a 37% reduction in circRNA nickingcompared to the T4 catalytic intron (FIG. 1I and FIG. 1J). Due toincreased splicing efficiency and intact circRNA output, the engineeredAnabaena PIE system proved to be overall superior to the engineered T4PIE system (FIG. 1J).

Internal homology between exon 2 and the GLuc coding sequence renderedthe optimized Anabaena PIE system incompatible with non-GLuc interveningregions. To adapt the circRNA construct for efficient circularization ofa variety of long intervening RNA sequences, a pair of spacer sequenceswere de novo designed based on the understanding of the parameters thataffect permuted catalytic group I intron splicing efficacy. These spacersequences were engineered with three priorities: 1) to be inert withregards to the folding of proximal intron and IRES structures; 2) tosufficiently separate intron and IRES secondary structures; and 3) tocontain a region of spacer-spacer complementarity to promote theformation of a ‘splicing bubble’ (FIG. 2A, Table 1). Homology arms atthe 5′ and 3′ ends of the precursor molecule were also included. Betweenthese sequences an EMCV IRES was inserted as well as coding regions forfive different proteins, including Gaussia luciferase, Fireflyluciferase, eGFP, human erythropoietin, and Cas9 endonuclease.Circularization of all five RNA sequences was achieved (FIG. 2B, Table1); circularization efficiency matched that of the stepwise-designedconstruct (FIG. 1J) and was highly reproducible between inserts but wasalso dependent on size, with long RNAs less efficiently circularized(FIG. 5A). In addition, it was found that long circRNAs were more proneto nicking in the presence of magnesium ions, resulting in accumulationof nicked circRNA during and after in vitro transcription and RNase Rdigestion which reduced the overall yields and the purity of the RNaseR-treated sample (FIG. 2B and FIG. 2C, FIG. 5A). RNase R did not fullydigest resistant Anabaena introns (FIG. 2B, bottom bands) or circularconcatenations (FIG. 2B, top bands).

It has been demonstrated that endogenous circRNA may produce smallquantities of protein (Legnini, I. et al., “Circ-ZNF609 Is a CircularRNA that Can Be Translated and Functions in Myogenesis,” Molecular Cell,66(1):22-37.e9 (2017)). As a means of assessing the ability ofengineered circRNAs to produce protein, RNase R-digested splicingreactions of each construct was transfected into human embryonic kidneycells (HEK293). Transfection of Gaussia or Firefly luciferase circRNAresulted in robust production of functional protein as measured byluminescence (FIG. 2D and FIG. 2G). Likewise, human erythropoietin wasdetected in cell culture media from transfection of erythropoietincircRNA, and EGFP fluorescence was observed from transfection of EGFPcircRNA (FIG. 2E and FIG. 2F). Co-transfection of Cas9 circRNA withsgRNA directed against GFP into HEK293 cells constitutively expressingGFP resulted in ablated fluorescence in up to 97% of cells in comparisonto an sgRNA-only control (FIG. 2H and FIG. 2I). Because RNase Rdigestion of splicing reactions is not always complete and precursor RNAcontains a functional IRES, a splice site deletion mutant of the GLucconstruct was created to measure the potential contribution ofimpurities to protein expression. When transfected at equal weightquantities to RNase-R digested splicing reactions, this splice sitedeletion mutant produced a barely detectable level of protein (FIG. 5Band FIG. 5C).

To establish exogenous circRNA as a reliable alternative to existinglinear mRNA technology it is desirable to maximize protein expression.Cap-independent translation mediated by an IRES can exhibit varyinglevels of efficiency depending on cell context and is generallyconsidered less efficient than cap-dependent translation when includedin bicistronic linear mRNA (Borman, A. M. et al., “Comparison ofPicornaviral IRES-Driven Internal Initiation of Translation in CulturedCells of Different Origins,” Nucleic Acids Research, 25(5):925-932(1997)). Similarly, the polyA tail stabilizes and improves translationinitiation efficiency in linear mRNA through the actions ofpolyadenylate binding proteins (Imataka, H., “A newly identifiedN-terminal amino acid sequence of human eIF4G binds poly(A)-bindingprotein and functions in poly(A)-dependent translation,” The EMBOJournal, 17.24:7480-489 (1998); Kahvejian, A. et al., “Mammalianpoly(A)-binding protein is a eukaryotic translation initiation factor,which acts via multiple mechanisms,” Genes & Development, 19(1):104-113(2005)). However, the efficiency of different IRES sequences and theinclusion of a polyA tract within the context of circRNA has not beeninvestigated. The EMCV IRES was replaced with 5′ UTR sequences fromseveral viral transcripts that contain known or putative IRESs, as wellas several other putative IRES sequences (Table 1, FIG. 6A)(Weingarten-Gabbay, S. et al., “Systematic discovery of cap-independenttranslation sequences in human and viral genomes,” Science, 351(6270)(2016)). It was found that the IRES from Coxsackievirus B3 (CVB3) was1.5-fold more effective than the commonly adopted EMCV IRES in HEK293cells (FIG. 3A). Because secondary structures proximal to the IRES,including within the coding region that directly follows the IRES, havethe potential to disrupt IRES folding and translation initiation,selected viral IRES sequences were tested in the context of Fireflyluciferase. While the CVB3 IRES was still superior to all others, theefficacy of several other IRESs, most notably the Poliovirus IRES, wasdramatically altered (FIG. 3B). The addition of an internal polyAsequence or a polyAC spacer control to IRES sequences was tested andshowed the ability to drive protein production above background levelsfrom engineered circRNA would alter protein expression. It was foundthat both sequences improved expression in all constructs, possibly dueto greater unstructured separation between the beginning of the IRESsequence and the exon-exon splice junction, which is predicted tomaintain stable structure (FIG. 3B, FIG. 6B). This greater degree ofunstructured separation may reduce steric hindrance occluding initiationfactor binding to IRES structures. In the case of EMCV and PoliovirusIRESs, polyA sequences improved expression beyond the improvement seenwith an unstructured polyAC spacer. This may suggest that theassociation of polyadenylate binding proteins may enhance IRESefficiency. After selecting the most effective polyA or polyAC constructfor each IRES, IRES efficacy in different cell types was explored,including human cervical adenocarcinoma (HeLa), human lung carcinoma(A549), and immortalized mouse pancreatic beta cells (Min6). jIRESefficacy varied depending on type, but the CVB3 IRES was superior in alltypes tested (FIG. 3C, FIG. 6C and FIG. 6D).

Purity of circRNA preparations is another factor essential formaximizing protein production from circRNA and for avoiding innatecellular immune responses. It has been shown that removal of dsRNA byHPLC eliminates immune activation and improves translation of linearnucleoside-modified IVT mRNA (Kariko, K. et al., “Generating the optimalmRNA for therapy: HPLC purification eliminates immune activation andimproves translation of nucleoside-modified, protein-encoding mRNA,”Nucleic Acids Research, 39(21):e142-e142 (2011)). However, no scalablemethods have been reported for purification of circRNA from byproductsof IVT and circularization reactions, which include dsRNA andtriphosphate-RNA that may engage RNA sensors and induce a cellularimmune response (Kariko, K. et al., “Generating the optimal mRNA fortherapy: HPLC purification eliminates immune activation and improvestranslation of nucleoside-modified, protein-encoding mRNA,” NucleicAcids Research, 39(21):e142-e142 (2011)). While the complete avoidanceof nicked circRNA was untenable due to mild degradation duringprocessing, substantially pure (90% circular, 10% nicked) circRNA wasobtained using gel extraction for small quantities and size exclusionHPLC for larger quantities of splicing reaction starting material (FIG.3D and FIG. 3E). In both cases, purification was followed with RNase Rtreatment to eliminate the majority of degraded RNA. When comparing theprotein expression of gel extracted or HPLC purified circRNA to RNase-Rdigested splicing reactions, HPLC purification was found to be asuperior method of purification that surpassed RNAse-R digestion alone(FIG. 3E and FIG. 3F).

It is unknown whether exogenous circRNA translation efficiency iscomparable to that of linear mRNA, and whether circRNA proteinproduction exhibits differences in stability. Using HPLC-purifiedengineered circRNA, the stability and efficacy of Gaussialuciferase-coding circRNA (CVB3-GLuc-pAC) was compared to equimolarquantities of a canonical unmodified 5′ methylguanosine-capped and 3′polyA-tailed linear GLuc mRNA as well as a commercially availablenucleoside modified linear GLuc mRNA (Trilink). Protein productionassessed by luminescence 24 hours post-transfection revealed thatcircRNA produced 811.2% more protein than unmodified linear mRNA at thisearly time point in HEK293 cells (FIG. 3H). Interestingly, circRNA alsoproduced 54.5% more protein than modified mRNA, demonstrating thatnucleoside modifications are not necessary for robust protein productionfrom circRNA. Similar results were obtained in HeLa cells (FIG. 3H).Luminescence data collected over six days showed that protein productionfrom circRNA was extended relative to that from linear mRNA in HEK293cells, with circRNA exhibiting a protein production half-life of 80hours, while the half-lives of protein production from unmodified andmodified linear mRNA were approximately 43 and 45 hours respectively(FIG. 3I). Due to increased expression or stability, circRNA alsoproduced substantially more protein than both unmodified and modifiedlinear mRNAs over its lifetime (FIG. 3J and FIG. 3K). In HeLa cells,circRNA exhibited a protein production half-life of 116 hours, while thehalf-lives of protein production from unmodified and modified linearmRNA were approximately 44 and 49 hours respectively (FIG. 3I). Thisagain resulted in substantially more protein production from circRNAover its lifetime compared to both unmodified and modified linear mRNAs(FIG. 3J and FIG. 3K).

Obtaining stable protein production from exogenous mRNA has been alongstanding goal of mRNA biotechnology. The possibility of adaptingcircular RNA for this purpose has been stifled by low circRNA productionefficiency, difficulty of purification, and weak protein expression.Indeed, these obstacles must be overcome before the stability of proteinproduction from circRNA can be fully assessed. The modular permutedgroup 1 catalytic intron-based system using a vector that includedhomology arms and spacers as described herein permits the efficientcircularization of a wide range of long RNAs. In addition, it was shownthat optimized circRNA is capable of producing large quantities ofprotein and that it can be effectively purified by HPLC. Finally, it wasshown that circRNA can produce greater quantities of protein for alonger duration than unmodified and modified linear RNA, providingevidence that circRNA holds potential as an alternative to mRNA for thestable expression of therapeutic proteins.

The results for FIG. 17 show that RNA circularization with a suboptimalconstruct can be promoted by increased temperature, but at the cost ofincreased degradation.

The results for FIGS. 18A-C show that circularization is sensitive toGTP concentration, insensitive to RNA concentration, and the two bandson the gel don't interchange (A, B, C respectively); this is also anexample of strong homology arm/5′ spacer circularization.

The results for FIG. 19 show that low protein expression fromunoptimized circRNA construct—no spacers, strong homology arms;demonstration of the importance of purification on circRNAexpression/stability.

The results for FIGS. 20A and B show that UTRs can improve expressionfrom circRNA. The results for FIG. 20C show that when used incombination with a 5′ spacer, the expression benefits of adding UTRsdisappear, suggesting that UTRs can act as spacers. The results for FIG.20D show that efficient circularization of UTR-containing constructs,all using the same spacers/homology arms.

The results for FIGS. 21A-G show expression assays using different IRESor spacer sequences. pA/UTR conditions in FIGS. E and F use the EMCVIRES and are able to improve expression to the level of the CVB3 IRES insome cases. FIG. G shows comparison of transfection reagent andnanoparticles; doesn't appear to be a difference here.

The results for FIGS. 22A and B show that addition of different 5′ or 3′spacers (in addition to an existing, designed spacer) can modulateexpression.

The results for FIGS. 23A and B show that introns can interfere withtranslation from different IRES sequences to different degrees; Anabaenainterferes less with CVB3/polioV IRESes, while T4 phage interferes lesswith EMCV. FIG. 23B shows in vivo assessment of different IRESes (mouseliver via LNP).

The results for FIG. 24 show that show that plasmids that promote thetranscription of circRNA precursor molecules in mammalian cells do notdemonstrate enhanced protein translation compared to plasmids thatpromote the transcription of the same circRNA precursor molecules withdeleted splice sites, suggesting that circularization does not occur inmammalian cells.

The results for FIGS. 25A and 25B show that circularization efficiencyof constructs containing a panel of IRES sequences with either agaussian or firefly luciferase coding region—efficiency is consistentdespite varying inserts. These constructs all have the same splicingsequences (defined as spacers, homology arms, and internal homology(which is part of the spacers.

The results for FIGS. 26A and 26B show that spiking in guanosinemonophosphate in the in vitro transcription reaction (in excess overguanosine triphosphate) reduces immunogenicity of circRNA preparations.Guanosine monophosphate is best used in combination with HPLC forsensitive cells.

Materials and Methods

Cloning and Mutagenesis

Protein coding, group I self-splicing intron, and IRES sequences werechemically synthesized (Integrated DNA Technologies) and cloned into aPCR-linearized plasmid vector containing a T7 RNA polymerase promoter byGibson assembly using a NEBuilder HiFi DNA Assembly kit (New EnglandBiolabs). Spacer regions, homology arms, and other minor alterationswere introduced using a Q5 Site Directed Mutagenesis Kit (New EnglandBiolabs).

circRNA Design, Synthesis, and Purification

RNA structure was predicted using RNAFold (Vicens, Q. et al., “Towardpredicting self-splicing and protein-facilitated splicing of group Iintrons,” RNA, 14(10):2013-2029 (2008)). Modified linear GLuc mRNA wasobtained from Trilink Biotechnologies. Unmodified linear mRNA or circRNAprecursors were synthesized by in-vitro transcription from a linearizedplasmid DNA template using a T7 High Yield RNA Synthesis Kit (NewEngland Biolabs). After in vitro transcription, reactions were treatedwith DNase I (New England Biolabs) for 20 minutes. After DNasetreatment, unmodified linear mRNA was column purified using a MEGAclearTranscription Clean-up kit (Ambion). RNA was then heated to 70° C. for 5minutes and immediately placed on ice for 3 minutes, after which the RNAwas capped using mRNA cap-2′-O-methyltransferase (NEB) and Vacciniacapping enzyme (NEB) according to the manufacturer's instructions.Polyadenosine tails were added to capped linear transcripts using E.coli PolyA Polymerase (NEB) according to manufacturer's instructions,and fully processed mRNA was column purified. For circRNA, after DNasetreatment additional GTP was added to a final concentration of 2 mM, andthen reactions were heated at 55° C. for 15 minutes. RNA was then columnpurified. In some cases, purified RNA was re-circularized: RNA washeated to 70° C. for 5 minutes and then immediately placed on ice for 3minutes, after which GTP was added to a final concentration of 2 mMalong with a buffer including magnesium (50 mM Tris-HCl, 10 mM MgCl2, 1mM DTT, pH 7.5; New England Biolabs). RNA was then heated to 55° C. for8 minutes, and then column purified. To enrich for circRNA, 20 μg of RNAwas diluted in water (86 uL final volume) and then heated at 65° C. for3 minutes and cooled on ice for 3 minutes. 20 U RNase R and 10 uL of 10×RNase R buffer (Epicenter) was added, and the reaction was incubated at37° C. for 15 minutes; an additional 10 U RNase R was added halfwaythrough the reaction. RNase R-digested RNA was column purified. RNA wasseparated on precast 2% E-gel EX agarose gels (Invitrogen) on the E-geliBase (Invitrogen) using the E-gel EX 1-2% program. Adequate circRNAseparation using other agarose gel systems was not obtained. Bands werevisualized using blue light transillumination and quantified usingImageJ. For gel extractions, bands corresponding to the circRNA wereexcised from the gel and then extracted using a Zymoclean Gel RNAExtraction Kit (Zymogen). For high-performance liquid chromatography, 30μg of RNA was heated at 65° C. for 3 minutes and then placed on ice for3 minutes. RNA was run through a 4.6×300 mm size-exclusion column withparticle size of 5 μm and pore size of 200 Å (Sepax Technologies; partnumber: 215980P-4630) on an Agilent 1100 Series HPLC (Agilent). RNA wasrun in RNase-free TE buffer (10 mM Tris, 1 mM EDTA, pH:6) at a flow rateof 0.3 mL/minute. RNA was detected by UV absorbance at 260 nm, but wascollected without UV detection. Resulting RNA fractions wereprecipitated with 5M ammonium acetate, resuspended in water, and then insome cases treated with RNase R as described above.

RNase H Nicking Analysis

Splicing reactions enriched for circRNA with RNase R and then columnpurified were heated at 65° C. for 5 minutes in the presence of a DNAprobe (Table 1) at five-fold molar excess, and then annealed at roomtemperature. Reactions were treated with RNase H (New England Biolabs)in the provided reaction buffer for 15 minutes at 37 C. RNA was columnpurified after digestion.

Reverse Transcription and cDNA Synthesis

For splice junction sequencing, splicing reactions enriched for circRNAwith RNase R and then column purified were heated at 65° C. for 5minutes and cooled on ice for 3 minutes to standardize secondarystructure. Reverse transcription reactions were carried out withSuperscript IV (Invitrogen) as recommended by the manufacturer using aprimer specific for a region internal to the putative circRNA. PCRproduct for sequencing was synthesized using Q5 polymerase (New EnglandBiolabs) and a pair of primers spanning the splice junction.

Tissue Culture and Transfections

HEK293, HEK293-GFP, HeLa, and A549 cells were cultured at 37° C. and 5%CO2 in Dulbecco's Modified Eagle's Medium (4500 mg/L glucose)supplemented with 10% heat-inactivated fetal bovine serum (hiFBS, Gibco)and penicillin/streptomycin. Min6 medium was additionally supplementedwith 5% hiFBS, 20 mM HEPES (Gibco) and 50 μM beta-mercaptoethanol(BioRad). Cells were passaged every 2-3 days. For all circRNA data setspresented in FIG. 2 except Cas9, 40-100 ng of RNase R-treated splicingreactions or HPLC-purified circRNAs were reverse transfected into 10,000HEK293 cells/100 uL per well of a 96-well plate using LipofectamineMessengerMax (Invitrogen) according to the manufacturer's instructions.For Cas9, 100 ng of in vitro transcribed sgRNA was reverse transfectedalone or cotransfected with 150 ng of RNase R-treated Cas9 splicingreaction into 50,000 HEK293-GFP cells/500 uL per well of a 24-well plateusing MessengerMax. For all RNA data sets presented in FIG. 3, equimolarquantities of each RNA were reverse transfected into 10,000 HEK293,HeLa, or A549 cells/100 uL per well of a 96-well plate usingMessengerMax. Min6 cells were transfected in 96-well plate formatbetween 60-80% confluency.

Protein Expression Analysis

For luminescence assays, cells and media were harvested 24 hourspost-transfection. To detect luminescence from Gaussia luciferase, 10-20uL of tissue culture medium was transferred to a flat-bottomedwhite-walled plate (Corning). 25 uL of BioLux Gaussia Luciferase reagentincluding stabilizer (New England Biolabs) was added to each sample andluminescence was measured on an Infinite 200Pro Microplate Reader(Tecan) after 45 seconds. To detect luminescence from Fireflyluciferase, 100 uL of Bright-Glo Luciferase reagent (Promega) was addedto each well, mixed, and incubated for 5 minutes. 100 uL of the culturemedium and luciferase reagent mix was then transferred to aflat-bottomed white-walled plate and luminescence was detected asdescribed above. GFP fluorescence was detected 24 hours aftertransfection and images were taken using an EVOS FL cell imager(Invitrogen). Erythropoietin was detected by solid phase sandwich ELISA(R&D Systems) essentially according to the manufacturer's instructionsexcept cell culture supernatant 24 hours post transfection was used, andsamples were diluted 1:200 before use.

Flow Cytometry

CRISPR-Cas9-mediated GFP ablation was detected by flow cytometry 96hours after transfection. HEK293-GFP and HEK293 control cells weretrypsinized and suspended in Dulbecco's Modified Eagle's Medium (4500mg/L glucose) supplemented with 10% fetal bovine serum andpenicillin/streptomycin. Cells were then washed twice in FACS buffer(PBS, 5% heat-inactivated fetal bovine serum) and resuspended in FACSbuffer containing Sytox Blue Dead Cell Stain (Thermo Fisher) accordingto the manufacturer's instructions, or FACS buffer alone for GFP andblank controls. Fluorescence was detected for 10,000 events on a BDFACSCelesta flow cytometer (BD Biosciences). Data was analyzed in Flowjo(Flowjo LLC).

Statistics

Statistical analysis of the results was performed by a two-tailedunpaired Welch's t-test, assuming unequal variances. Differences wereconsidered significant when p<0.05. Statistical details of individualexperiments are present in figure legends.

Example 2

Circular RNAs (circRNAs) are a class of single-stranded RNAs with acontiguous structure that have enhanced stability and a lack of endmotifs necessary for interaction with various cellular proteins. Here,it is shown that unmodified exogenous circRNA is able to bypass cellularRNA sensors and thereby avoid provoking an immune response in RIG-I andtoll-like receptor (TLR) competent cells and in mice. The immunogenicityand protein expression stability of circRNA preparations is found to bedependent on purity, with small amounts of contaminating linear RNAleading to robust cellular immune responses. Unmodified circRNA is lessimmunogenic than unmodified linear mRNA in vitro, in part due to evasionof TLR sensing, and provokes a cytokine response that is similar to thatinduced by uridine-modified linear mRNA. Additionally, it was found thaturidine modification of circRNA disrupts internal ribosome entry site(IRES)-mediated translation and does not have a significant effect oncytokine response. Finally, the data shows the first demonstration ofexogenous circRNA delivery and translation in vivo, and the data showsthat circRNA translation is extended in adipose tissue in comparison tounmodified and uridine-modified linear mRNAs.

INTRODUCTION

CircRNAs are a class of RNAs with a range of protein-coding andnon-coding functions (Legnini, I. et al. Circ-ZNF609 Is a Circular RNAthat Can Be Translated and Functions in Myogenesis. Mol. Cell 66,22-37.e9 (2017); Li, Z. et al. Exon-intron circular RNAs regulatetranscription in the nucleus. Nat. Struct. Mol. Biol. 22, 256-264(2015); Hansen, T. B. et al. Natural RNA circles function as efficientmicroRNA sponges. Nature 495, 384-388 (2013); and Barrett, S. P. &Salzman, J. Circular RNAs: analysis, expression and potential functions.Development 143, 1838-1847 (2016). Eukaryotic cells generate circRNAsthrough backsplicing, while the genomes of viral pathogens such ashepatitis D virus and plant viroids can also be circular (Chen, L.-L. &Yang, L. Regulation of circRNA biogenesis. RNA Biol. 12, 381-388 (2015);Jeck, W. R. & Sharpless, N. E. Detecting and characterizing circularRNAs. Nat. Biotechnol. 32, 453-461 (2014); Wang, Y. & Wang, Z. Efficientbacksplicing produces translatable circular mRNAs. RNA 21, 172-179(2014); Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J. &Kleinschmidt, A. K. Viroids are single-stranded covalently closedcircular RNA molecules existing as highly base-paired rod-likestructures. Proc. Natl. Acad. Sci. U.S.A. 73, 3852-3856 (1976); Kos, A.,Dijkema, R., Arnberg, A. C., van der Meide, P. H. & Schellekens, H. Thehepatitis delta (delta) virus possesses a circular RNA. Nature 323,558-560 (1986); Chen, Y. G. et al. Sensing Self and Foreign CircularRNAs by Intron Identity. Mol. Cell 67, 228-238.e5 (2017)). It hasrecently been proposed that cells have evolved a splicing-dependentmechanism for the discrimination of endogenous and exogenous circRNA,using RIG-1 as a cytoplasmic sensor of exogenous circRNA (Chen, Y. G. etal. Sensing Self and Foreign Circular RNAs by Intron Identity. Mol. Cell67, 228-238.e5 (2017)). While circRNA does not contain the triphosphatemotif canonically required for RIG-I activation, it has been suggestedthat RIG-I may transiently interact with circRNA devoid of host nuclearproteins, leading to a canonical RIG-I mediated antiviral response(Chen, Y. G. et al. Sensing Self and Foreign Circular RNAs by IntronIdentity. Mol. Cell 67, 228-238.e5 (2017); Loo, Y. M. & Gale, M., Jr.Immune signaling by RIG-I-like receptors.—PubMed—NCBI. Available at:https://www.ncbi.nlm.nih.gov/pubmed/21616437. (Accessed: 7 May 2018)).However, the mechanism of RIG-I-mediated recognition of circRNA remainsunclear. In addition to RIG-I, it is also possible that circRNAinteracts with other RNA sensors such as the endosomal TLRs 3, 7 and 8,which have been shown to activate signaling in response to linear ssRNAand dsRNA motifs as well as RNA degradation products such as uridine andguanosine-uridine rich fragments (Tanji, H. et al. Toll-like receptor 8senses degradation products of single-stranded RNA. Nat. Struct. Mol.Biol. 22, 109-115 (2015); Zhang, Z. et al. Structural Analysis Revealsthat Toll-like Receptor 7 Is a Dual Receptor for Guanosine andSingle-Stranded RNA. Immunity 45, 737-748 (2016); Bell, J. K., Askins,J., Hall, P. R., Davies, D. R. & Segal, D. M. The dsRNA binding site ofhuman Toll-like receptor 3. Proc. Natl. Acad. Sci. U.S.A. 103, 8792-8797(2006); and Tatematsu, M., Nishikawa, F., Seya, T. & Matsumoto, M.Toll-like receptor 3 recognizes incomplete stem structures insingle-stranded viral RNA. Nat. Commun. 4, 1833 (2013)). To reduce aninnate cellular immune response to exogenous RNA, nucleosidemodifications such as pseudouridine (ψ), N¹-methylpseudouridine (m1ψ),and 5-methoxyuridine (5moU) have been developed for use in linear mRNA(Svitkin, Y. V. et al. N1-methyl-pseudouridine in mRNA enhancestranslation through eIF2α-dependent and independent mechanisms byincreasing ribosome density. (Nucleic Acids Res. 45, 6023-6036 (2017);Karikó, K., Muramatsu, H., Ludwig, J. & Weissman, D. Generating theoptimal mRNA for therapy: HPLC purification eliminates immune activationand improves translation of nucleoside-modified, protein-encoding mRNA.Nucleic Acids Res. 39, e142 (2011); Karikó, K. et al. Incorporation ofPseudouridine Into mRNA Yields Superior Nonimmunogenic With IncreasedTranslational Capacity and Biological Stability. Mol. Ther. 16, 1833(2008)). These modifications have been shown to prevent linear mRNA fromactivating TLRs and RIG-I (Karikó, K., Buckstein, M., Ni, H. & Weissman,D. Suppression of RNA recognition by Toll-like receptors: the impact ofnucleoside modification and the evolutionary origin of RNA. Immunity 23,165-175 (2005); Durbin, A. F., Wang, C., Marcotrigiano, J. & Gehrke, L.RNAs Containing Modified Nucleotides Fail To Trigger RIG-IConformational Changes for Innate Immune Signaling. MBio 7, (2016)). RNAmodification with N⁶-methyladenosine (m6A) has been shown to mediatecap-independent translation in endogenous linear and circRNAs (Meyer etal. 2015; Yang et al. 2017). The contribution of TLRs to circRNAimmunogenicity, and the effects of nucleoside modifications on exogenouscircRNA translation, stability, and immunogenicity, have yet to bereported.

Recently, circRNA was developed for stable protein production inmammalian cells (Wesselhoeft, R. A., Kowalski, P. S. & Anderson, D. G.Engineering circular RNA for potent and stable translation in eukaryoticcells. Nat. Commun. 9, 2629 (2018)). As described herein, theimmunogenicity and translatability of exogenous circRNA in vitro and invivo was investigated to determine the potential utility of circRNA forprotein production applications. It was demonstrated herein thatexogenous circRNA does not stimulate a cellular immune response in RIG-Iand TLR competent cells. Additionally, it is shown that unlike linearmRNA, IRES-dependent circRNA does not benefit from modification with m1ψin terms of protein expression and immunogenicity or modification withm6A in terms of protein expression. It was found that circRNA iscompatible with lipid nanoparticle-mediated delivery and is effectivelytranslated in vivo without provoking an RNA-mediated innate immuneresponse, while protein expression from circRNA exhibits greaterstability that that from uridine-modified linear mRNA in adipose tissue.

Results

Purification of Exogenous circRNA Ablates Immunogenicity

Using the optimized permuted intron-exon (PIE) splicing methodpreviously reported, circRNA precursors were synthesized containing acoxsackievirus B3 internal ribosome entry site (CVB3 IRES), a Gaussialuciferase (GLuc) message, two designed spacer sequences, two shortregions corresponding to exon fragments of the PIE construct, and the 3′and 5′ intron segments of the permuted Anabaena pre-tRNA group I intronby run-off transcription (FIG. 7A-B) (Wesselhoeft, R. A., Kowalski, P.S. & Anderson, D. G. Engineering circular RNA for potent and stabletranslation in eukaryotic cells. Nat. Commun. 9, 2629 (2018); Puttaraju,M. & Been, M. Group I permuted intron-exon (PIE) sequences self-spliceto produce circular exons. Nucleic Acids Res. 20, 5357-5364 (1992)). Inthe presence of GTP and Mg²⁺, these precursor RNA molecules undergo thedouble transesterification reactions characteristic of group I catalyticintrons, but because the exons are already fused, the region between thetwo intron segments is excised as a covalently 5′ to 3′ linked circle(FIG. 7A) (Puttaraju, M. & Been, M. Group I permuted intron-exon (PIE)sequences self-splice to produce circular exons. Nucleic Acids Res. 20,5357-5364 (1992)). To confirm that circular products were obtained, thesplicing reaction was treated with RNase R, a 3′ to 5′ RNA exonuclease,and observed enrichment of the putative circRNA band (FIG. 7C) (Suzuki,H. et al. Characterization of RNase R-digested cellular RNA source thatconsists of lariat and circular RNAs from pre-mRNA splicing. NucleicAcids Res. 34, e63-e63 (2006)). Subsequent purification of the RNaseR-treated splicing reaction by HPLC and then digestion witholigonucleotide-targeted RNase H produced a single major band incontrast to two major bands yielded by RNase H-digested linear precursorRNA that contains all of the same sequence elements as the circRNAprecursor with the exception of the splice site nucleotides (FIG. 7C,ΔS), confirming circularity. Splicing reactions containing circRNAdemonstrated improved protein production and expression stability ofprotein production in comparison to polyadenylated andphosphatase-treated linear precursor after transfection into 293 cells(FIG. 7. G-I).

To probe the immunogenicity of circRNA, two cell lines (human embryonickidney, 293; human lung carcinoma, A549) were selected that had beenobserved to elicit differential cell viability and GLuc expressionstability responses upon transfection of unpurified circRNA splicingreactions (FIGS. 7E and F). After the circularization protocol, thesesplicing reactions are expected to contain circRNA, excisedtriphosphorylated introns, linear and circular concatenations, anddegradation products of both linear RNA and circRNA, some of which istriphosphorylated. While the splicing reaction proceeds nearly tocompletion under the circularization conditions used herein, sometriphosphorylated linear precursor RNA is also present. Several stepswere then applied of purification to the unpurified splicing reactionsand confirmed circRNA enrichment by gel electrophoresis: RNase R toenrich circRNA, HPLC to remove non-circular components, and phosphataseto remove residual triphosphates (FIG. 7D). To determine the extent ofthe innate cellular immune response to transfected RNA, the release of awide range of cytokines and chemokines into the culture medium wasmonitored, as well as the protein expression stability from circRNA andcell viability.

It was found that RNase R digestion of splicing reactions wasinsufficient to ablate cytokine release in A549 cells in comparison tountransfected controls (FIG. 7F, FIG. 8G). The addition of HPLCpurification was furthermore insufficient to ablate cytokine release,although we did note a significant reduction in IL-6 and a significantincrease in IFN-α1 compared to the unpurified splicing reaction,suggesting that combined RNase R and HPLC may have depleted someimmunogenic RNA species while enriching others (FIG. 7F). The additionof HPLC purification was furthermore insufficient to ablate cytokinerelease, although a reduction in IL-6 compared to the unpurifiedsplicing reaction (FIG. 7F) was noted. Interestingly, the addition of aphosphatase treatment after HPLC purification and before RNase Rdigestion dramatically reduced the expression of all upregulatedcytokines that we assessed in A549 cells, with secreted MCP1, IL-6,IFN-α1, TNF-α, and IP-10 falling to undetectable oruntransfected-baseline levels (FIG. 7F). Substantial cytokine release in293 cells was not observed, consistent with the observation that the3-day protein expression stability phenotype of these cells isrelatively unaffected by the degree of circRNA purity and previousreports indicating that 293 cells do not express several key RNA sensors(FIG. 7E, FIG. 8G) (Hornung, V. et al. Quantitative expression oftoll-like receptor 1-10 mRNA in cellular subsets of human peripheralblood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J.Immunol. 168, 4531-4537 (2002)). In contrast, increased circRNA purityimproved GLuc expression stability in transfected A549 cells, withcompletely purified circRNA demonstrating a stability phenotype similarto that of transfected 293 cells (FIGS. 7E and F). Likewise, a trend ofincreased circRNA purity improved A549 cell viability 3 dayspost-transfection while 293 cell viabilities remained largelyunaffected, consistent with a lack of inflammatory signaling in 293cells and diminishing inflammatory signaling in A549 cells withincreasing circRNA purity (FIGS. 7E and F). Together, these resultsdemonstrate that circRNA purity strongly affects its immunogenicpotential, and that fully purified circRNA is significantly lessimmunogenic than unpurified or incompletely purified splicing reactions.The stability of protein production from circRNA is also dependent oncircRNA purity and the sensitivity of transfected cell types tocontaminating RNA species. A time course experiment monitoring RIG-I,IFN-β1, IL-6 and RANTES transcript induction within the first 8 hoursafter transfection of A549 cells with splicing reactions or fullypurified circRNA did not reveal a transient response to circRNA (FigureS2F). Purified circRNA similarly failed to induce pro-inflammatorytranscripts in RAW264.7 murine macrophages (Figure S2G). To generalizethese findings to another synthetic circRNA construct, we tested theinduction of pro-inflammatory transcripts in response to transfection ofA549 cells with purified circRNA containing an EMCV IRES and EGFP codingregion, and again failed to observe substantial induction (Figure S2H).These data demonstrate that non-circular components of the splicingreaction are responsible for the immunogenicity observed in previousstudies and that circRNA is not a natural ligand for RIG-I.

Non-Circular Components of the Splicing Reaction Contribute toImmunogenicity

To explore the source of immunogenicity in circRNA splicing reactions,each component of the splicing reaction was purified by HPLC andassessed cytokine release and cell viability upon transfection of A549cells (FIGS. 8A and B). Because there was difficulty obtaining suitablypure linear precursor RNA from the splicing reaction, precursor RNA inthe form of the splice site deletion mutant (ΔS) (FIG. 8B, bottom right)was separately synthesized and purified. Additionally, the circRNA peakwas split into two fractions to control for nicked RNA peak overlap(FIG. 8B). Robust IL-6, RANTES, and IP-10 release was observed inresponse to most species present within the splicing reaction as well asprecursor RNA (FIG. 8C, FIG. 9G). Early circRNA fractions elicitedcytokine responses comparable to other non-circRNA fractions, indicatingthat even relatively small quantities of linear RNA contaminants areable to induce a substantial cellular immune response in A549 cells.Late circRNA fractions elicited no cytokine response in excess of thatfrom untransfected controls. Consistent with cytokine releaseobservations, A549 cell viability 36 hours post transfection wassignificantly greater for late circRNA fractions compared to all otherfractions (FIG. 8D).

Because it has been previously reported that circRNA may induce RIG-Itranscription in a self-regulatory feedback loop, RIG-I and IFN-β1transcript induction was analyzed upon transfection of A549 cells withlate circRNA HPLC fractions (Chen, Y. G. et al. Sensing Self and ForeignCircular RNAs by Intron Identity. Mol. Cell 67, 228-238.e5 (2017)). Asignificantly weaker induction of both RIG-I and IFN-β1 transcripts forlate circRNA fractions was observed in comparison with precursor RNA andunpurified splicing reactions (FIG. 8E). Furthermore, it was found thatRNase R treatment of splicing reactions alone was not sufficient toablate this effect (FIG. 8F), while contamination of purified circRNAwith very small quantities of the RIG-I ligand 3p-hpRNA inducedsubstantial RIG-I transcription (FIG. 9I). In HeLa cells, transfectionof RNase R-digested splicing reactions, but not purified circRNA,induced RIG-I and IFN-β1, although it was found that HeLa cells to beless sensitive than A549 cells to contaminating RNA species (FIG. 9L).These data suggest that non-circular components of the splicing reactionare responsible for the immunogenicity observed in previous studies andthat circRNA is not an endogenous ligand for RIG-I.

Nucleoside Modification of circRNA is Disruptive

Nucleoside modifications such as 5-methylcytidine (m5C),N6-methyladenosine (m6A), and pseudouridine (ψ) have been reported todecrease the immunogenicity of linear mRNA in vitro and in some contextsin vivo by preventing ribonucleotides from interacting with cellular RNAsensors such as the endosomal TLRs 3, 7, and 8 and RIG-I (Karikó, K.,Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition byToll-like receptors: the impact of nucleoside modification and theevolutionary origin of RNA. Immunity 23, 165-175 (2005); Durbin, A. F.,Wang, C., Marcotrigiano, J. & Gehrke, L. RNAs Containing ModifiedNucleotides Fail To Trigger RIG-I Conformational Changes for InnateImmune Signaling. MBio 7, (2016)). N6-methyladenosine (m6A) has beenreported to mediate internal ribosome entry and translation on linearRNAs and separately on endogenous circRNAs (Meyer et al. 2015; Yang etal. 2017). The effects of these modifications on the utility of mRNA invivo may be variable however, as ψ-mRNA delivered to the liver does notreduce immunogenicity or improve protein production (Kauffman, K. J. etal. Efficacy and immunogenicity of unmodified and pseudouridine-modifiedmRNA delivered systemically with lipid nanoparticles in vivo.Biomaterials 109, 78-87 (2016)). Recently, it was reported thatincorporation of m1ψ diminishes mRNA immunogenicity and improves proteinexpression to a greater degree than incorporation of ψ (Svitkin, Y. V.et al. N1-methyl-pseudouridine in mRNA enhances translation througheIF2α-dependent and independent mechanisms by increasing ribosomedensity. Nucleic Acids Res. 45, 6023-6036 (2017) and Andries, O. et al.N(1)-methylpseudouridine-incorporated mRNA outperformspseudouridine-incorporated mRNA by providing enhanced protein expressionand reduced immunogenicity in mammalian cell lines and mice. J. Control.Release 217, 337-344 (2015)). The effects of nucleoside modifications oncircRNA translation efficiency and immunogenicity have not been tested.Because of previous difficulties with circRNA purification, theimmunogenicity of purified circRNA relative to that of unmodified linearmRNA has also not been assessed. Therefore, it was sought to evaluatethe GLuc protein expression stability and cytokine release profile ofpurified unmodified and m1ψ-modified circRNA in comparison to unmodifiedand m1ψ-modified linear mRNA in A549 and 293 cells (FIG. 9A).

Initial attempts to circularize m1ψ-circRNA using the PIE method wereunsuccessful, as complete replacement of uridine with m1ψ in PIEconstruct precursors abolished ribozyme activity while partialreplacement dramatically reduced splicing efficiency (FIG. 9B). Analternative method of circRNA preparation using T4 RNA ligase I andsplint oligonucleotides designed to bring the ends of the precursor RNAinto proximity for ligation (FIG. 10G) (Sonja Petkovic, S. M. RNAcircularization strategies in vivo and in vitro. Nucleic Acids Res. 43,2454 (2015)). Using optimized splint oligonucleotides and annealingconditions, 40% circularization efficiency of the 1.5 kb precursor RNAwas obtained (FIGS. 10H and I). Complete replacement of uridine with m1ψdid not impede circularization using this method and fully modifiedcircular products were obtained (FIG. 9C).

Upon transfection of 293 and A549 cells with m1ψ-circRNA, no proteinexpression was observed, and thus the stability of protein expressionfrom modified circRNA was not determined (FIG. 9D). Unmodified circRNAdisplayed enhanced protein expression stability in HEK293 and A549 cellscompared to both unmodified and modified linear mRNA (FIGS. 9E and F).Interestingly, it was found that unmodified linear mRNA provoked agreater cytokine response than unmodified circRNA in immunoresponsiveA549 cells despite capping, phosphatase treatment, and HPLC purificationto remove RIG-I ligands. In contrast, both m1ψ-circRNA and m1ψ-mRNA didnot significantly alter cytokine release profiles (FIG. 9F, FIG. 11E).A549 cell viability was diminished upon transfection of unmodifiedlinear mRNA, but not unmodified circRNA or either m1ψ-RNAs (FIG. 9F).Consistent with data from FIGS. 7A-F, significant differences in 293cytokine release at 24 hours post-transfection and cell viability at 3days post-transfection was not detected (FIG. 9E, FIG. 11E). Theseexperiments indicate that circRNA is less immunogenic than capped andpolyadenylated linear mRNA and that nucleoside modification of circRNAis unnecessary for protection against innate immune sensors.

CircRNA Evades Detection by Toll-Like Receptors

Because capped and polyadenylated linear mRNA was able to triggercytokine secretion while circRNA did not, the ability of different RNAsto activate TLRs in reporter cell lines was investigated. TLRs 3, 7, and8 are known to detect RNA in endosomes and initiate an inflammatorycascade (Takumi Kawasaki, T. K. Toll-Like Receptor Signaling Pathways.Front. Immunol. 5, (2014)). TLR3 binds to dsRNA and stem structures inviral ssRNA, while TLR7 and human TLR8 bind to ssRNA and nucleosidedegradation products (guanosine for TLR7 and uridine for TLR8), withboth ligands necessary for TLR activation (Tanji, H. et al. Toll-likereceptor 8 senses degradation products of single-stranded RNA. Nat.Struct. Mol. Biol. 22, 109-115 (2015); Zhang, Z. et al. StructuralAnalysis Reveals that Toll-like Receptor 7 Is a Dual Receptor forGuanosine and Single-Stranded RNA. Immunity 45, 737-748 (2016); Bell, J.K., Askins, J., Hall, P. R., Davies, D. R. & Segal, D. M. The dsRNAbinding site of human Toll-like receptor 3. Proc. Natl. Acad. Sci.U.S.A. 103, 8792-8797 (2006); and Tatematsu, M., Nishikawa, F., Seya, T.& Matsumoto, M. Toll-like receptor 3 recognizes incomplete stemstructures in single-stranded viral RNA. Nat. Commun. 4, 1833 (2013)).To control for structural and sequence differences between linear andcircular RNAs, a linearized version of the circRNA was constructed. Thisconstruct contained all of the components of the spliced circRNA, andwas created by deleting the intron and homology arm sequences(linearized RNA, FIG. 10A, FIG. 12G). All linearized RNAs wereadditionally treated with phosphatase (in the case of capped RNAs, aftercapping) and purified by HPLC. While a response to linearized orcircular RNA in TLR7 reporter cells was not found, both TLR3 and TLR8reporter cells were activated by capped linearized RNA, polyadenylatedlinearized RNA, the nicked circRNA fraction, and the early circRNAfraction (FIG. 10B). Interestingly, the late circRNA fraction did notprovoke a TLR-mediated response in any cell line, similarly to m1ψ-mRNA(FIG. 10B). However, the addition of uridine, but not cytidine, to themedia of TLR8 reporter cells transfected with circRNA partially revertedthis effect and resulted in SEAP secretion, indicating thattrans-addition of one of the two RNA degradation signals needed for TLR8activation can compensate for the lack of circRNA detection by TLR8(FIG. 10C, FIG. 12H).

Next, purified circRNA was linearized using two methods: treatment ofcircRNA with heat in the presence of magnesium ions, and DNAoligonucleotide-guided RNase H digestion (FIG. 10D). Both methodsyielded a majority of full-length linear RNA with small amounts ofintact circRNA, although heat treatment resulted in a greater proportionof lower molecular weight linear RNA degradation products (FIG. 10E).Transfection of circRNA degraded by both heat and RNase H prompted SEAPsecretion in TLR8 reporter cells (FIG. 4F). No activation was observedin TLR3 and TLR7 reporter cells for degraded or intact conditionsdespite activation of TLR3 by in vitro transcribed linearized RNA (FIG.4F, FIG. 12I). These results indicate that circRNA is able to avoiddetection by TLRs, and that TLR8 evasion is a result of circularconformation.

Exogenous circRNA is Translatable In Vivo

Translation and immunogenicity of unmodified and m1ψ-modified humanerythropoietin (hEpo) linear mRNAs and circRNAs was first examined, withlinear mRNAs identical to those depicted in FIG. 9A with the exceptionof the coding region (FIG. 13, FIG. 14A). Equimolar transfection ofm1ψ-mRNA and unmodified circRNA resulted in robust protein expression in293 cells (FIG. 14B). hEpo linear mRNA and circRNA displayed similarrelative protein expression patterns and cell viabilities in comparisonto GLuc linear mRNA and circRNA upon equal weight transfection of 293and A549 cells (FIGS. 14C and D). In mice, hEpo was detected in serumafter injection of hEpo circRNA or linear mRNA into visceral adipose(FIGS. 11A and D). hEpo detected after injection of unmodified circRNAdecayed more slowly than that from unmodified or m1ψ-mRNA and was stillpresent 42 hours post injection (FIG. 11B). A rapid decline in serumhEpo upon injection of unpurified circRNA splicing reactions orunmodified linear mRNA (FIG. 11B) was observed. Injection of unpurifiedsplicing reactions furthermore produced a cytokine response detectablein serum that was not observe for the other RNAs, including purifiedcircRNA (FIG. 11C, FIG. 15).

CircRNA is Compatible with Lipid Nanoparticles

Lipid nanoparticles have shown significant potential for use as deliveryvehicles for therapeutic RNAs, including the delivery of mRNA to tissues(Oberli, M. A. et al. Lipid Nanoparticle Assisted mRNA Delivery forPotent Cancer Immunotherapy. Nano Lett. 17, 1326-1335 (2017); YanezArteta, M. et al. Successful reprogramming of cellular proteinproduction through mRNA delivered by functionalized lipid nanoparticles.Proc. Natl. Acad. Sci. U.S.A. 115, E3351-E3360 (2018); and Kaczmarek, J.C., Kowalski, P. S. & Anderson, D. G. Advances in the delivery of RNAtherapeutics: from concept to clinical reality. Genome Med. 9, 60(2017)). To assess the efficacy of lipid nanoparticles for circRNAdelivery in vivo, purified circRNA was formulated into nanoparticleswith the ionizable lipidoid cKK-E12 (Dong, Y. et al. Lipopeptidenanoparticles for potent and selective siRNA delivery in rodents andnonhuman primates. Proc. Natl. Acad. Sci. U.S.A. 111, 3955-3960 (2014);and Kauffman, K. J. et al. Optimization of Lipid NanoparticleFormulations for mRNA Delivery in Vivo with Fractional Factorial andDefinitive Screening Designs. Nano Lett. 15, 7300-7306 (2015)). Theseparticles formed uniform multilamellar structures with an average size,polydispersity index, and encapsulation efficiency similar to that ofparticles containing commercially available control linear mRNA modifiedwith 5-methoxyuridine (5moU, FIG. 12A, Table 2). Purified hEpo circRNAencapsulated in LNPs displayed robust expression upon addition to 293cells in comparison to 5moU-mRNA (FIG. 12B, left). This commerciallyavailable mRNA performed similarly to the m1ψ-mRNA that was usedpreviously relative to circRNA (FIG. 14C). Protein expression stabilityfrom LNP-RNA in 293 cells was similar to that from RNA delivered bytransfection reagent with the exception of a slight delay in decay forboth 5moU-mRNA and circRNA (FIG. 12B, right). Encapsulation in LNPs didnot alter RIG-I/IFN-β1 induction or TLR activation in vitro, withunmodified circRNA failing to activate immune sensors in a mannersimilar to 5moU-mRNA (FIGS. 12C and D).

In mice, LNP-RNA was locally injected into visceral adipose tissue (FIG.16B). Serum hEPo expression from circRNA was lower but comparable withthat from 5moU-mRNA 6 hours after injection of LNP-RNAs into visceraladipose or intravenous delivery to liver (FIG. 12E, FIG. 16E). SerumhEpo detected after adipose injection of unmodified LNP-circRNA decayedmore slowly than that from LNP-5moU-mRNA, with a delay in expressiondecay present in serum similar to that noted in vitro (FIG. 12F);however, serum hEpo detected after intravenous injection of LNP-circRNAdecayed at the same rate as that from LNP-5moU-mRNA (FIG. 16E). Anincrease in serum cytokines was not observed, or local RIG-I, TNF-α, orIL-6 transcript induction after injection of LNP-5moU-mRNA orLNP-circRNA (FIGS. 16C and D).

TABLE 2 Ckk-E12 Size Intensity Encapsulation Formulation PolydispersityMean (nm) Efficacy (%) 5moU-mRNA 0.14 ± 0.02 92 ± 6 75 ± 6 Unpurified0.13 ± 0.04 87 ± 7 75 ± 13 Circular 0.12 ± 0.03 95 ± 7 77 ± 14Physicochemical properties of LNP-RNAs (data presented as mean±SD, n=3).

Discussion

In this work it was demonstrated that exogenous circRNA evades RNAsensors and that expression is extended relative to linear mRNAfollowing injection into mouse adipose tissue. While previous studiesexamining circRNA immunogenicity have proposed that exogenous circRNAprovokes a strong innate cellular immune response mediated by RIG-I, dueto an absence of associated host splicing factors (Chen et al. 2017), itwas found in this study that circRNA does not activate several knowncellular RNA sensors including TLRs and RIG-I (Chen, Y. G. et al.Sensing Self and Foreign Circular RNAs by Intron Identity. Mol. Cell 67,228-238.e5 (2017)). These discordant results are likely to be the resultof impurities in circRNA preparations. Previous studies have usedcircRNA purified by RNase R (Chen, Y. G. et al. Sensing Self and ForeignCircular RNAs by Intron Identity. Mol. Cell 67, 228-238.e5 (2017)). Thisstudy found that treatment with RNase R is not sufficient to obtain purecircRNA and enriches multiple resistant RNA species, which includecircRNA and linear RNAs with structured 3′ ends. Furthermore, even smallquantities of contaminating linear RNA, some of which may harbortriphosphates and may be present after HPLC purification, are sufficientto provoke robust cellular immune responses (FIG. 9C). HPLC purificationof circRNA presents unique difficulties, as nicked circRNA and intactcircRNA are equal in molecular weight and their respective peaks partlyoverlap. Degradation products of triphosphorylated precursor RNA willalso separate within the circRNA peak, and therefore gentle circRNApreparation is required. Phosphatase treatment, minimizing heat exposurein the presence of divalent cations, and stringent HPLC peak selectioncan reduce these hazards. Using the purification protocol describedhere, it was found that circRNA does not elicit substantial innateimmune responses from TLR and RIG-I competent cells, in contrast toother components of the splicing reaction, or from mouse adipose tissue,despite the absence of circRNA-associated host splicing factors. Usingthe purification protocol described here, it was found that circRNA doesnot elicit substantial innate immune responses from TLR and RIG-Icompetent cells, in contrast to other components of the splicingreaction, or from mouse adipose tissue. In addition, protein productionfrom purified circRNA is significantly more stable than that fromunpurified circRNA and transfection of purified circRNA results ingreatly improved cell viability (FIG. 7F, FIG. 8D), both of which areindicators of an antiviral response resulting from non-circularcontaminants (Loo, Y. M. & Gale, M., Jr. Immune signaling by RIG-I-likereceptors.—PubMed—NCBI. Available at:https://www.ncbi.nlm.nih.gov/pubmed/21616437. (Accessed: 7 May 2018)).Nucleoside modifications, especially uridine modifications, have beenreported to reduce linear mRNA immunogenicity by preventing detection byRNA sensors, which may be important for RNA function in some tissuetypes (Karikó, K., Buckstein, M., Ni, H. & Weissman, D. Suppression ofRNA recognition by Toll-like receptors: the impact of nucleosidemodification and the evolutionary origin of RNA. Immunity 23, 165-175(2005); Durbin, A. F., Wang, C., Marcotrigiano, J. & Gehrke, L. RNAsContaining Modified Nucleotides Fail To Trigger RIG-I ConformationalChanges for Innate Immune Signaling. MBio 7, (2016)). With theconstructs described here, enhanced protein expression stability fromm1ψ-mRNA compared to unmodified mRNA in vitro and in adipose tissue wasobserved (FIGS. 9E,11B). This may be a secondary outcome of immuneevasion or an unrelated primary effect of modification. Modification ofcircRNA precursor molecules with m1ψ interfered with splicing in the PIEconstructs and translation in the enzymatically circularized RNAs,suggesting that m1ψ significantly changes the folding of ribozyme andIRES structures.

Modification of RNA with m6A has been shown to promote cap-independenttranslation of endogenous linear and circular RNAs in living cells andexogenous linear RNAs in cell lysates (Meyer et al. 2015; Yang et al.2017). We found that partial replacement of adenosine with m6A was notsufficient to drive translation from exogenous intact or linearizedcircRNA in living cells, consistent with previous reports indicating theinvolvement of nuclear RNA binding proteins in assisting m6A-dependenttranslation (FIGS. 9F,G) (Lin et al. 2016).

Unlike linear mRNA, circRNA relies heavily on folded RNA structures,including the permuted group I intron and IRES, for splicing andtranslation. Modification of circRNA precursor molecules with m1ψ andm6A interfered with splicing in the PIE constructs and translation inthe enzymatically circularized RNAs, suggesting that modificationssignificantly change the folding of these structural elements (FIGS.9B,D,F,G).

Incorporation of ψ has been shown to enhance base-stacking interactions,which may lead to structural alterations; however, it is possible thatother nucleoside modifications may be more compatible with ribozyme andIRES structures and allow for the study of modified circRNA translationduring stability (Davis, D. R. Stabilization of RNA stacking bypseudouridine. Nucleic Acids Res. 23, 5020 (1995)). While it is knownthat modified linear mRNA is able to avoid detection by TLRs, it wassurprising to discover that unmodified circRNA exhibits the sameproperty. Recently, the ligands of TLR7 and TLR8 have been reported asdegradation products of RNA including short stretches of ssRNA andnucleosides (Tanji, H. et al. Toll-like receptor 8 senses degradationproducts of single-stranded RNA. Nat. Struct. Mol. Biol. 22, 109-115(2015); Zhang, Z. et al. Structural Analysis Reveals that Toll-likeReceptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA.Immunity 45, 737-748 (2016)). These degradation products are presumablyproduced by nucleases in the endosome shortly after the RNA isinternalized (Roers, A., Hiller, B. & Hornung, V. Recognition ofEndogenous Nucleic Acids by the Innate Immune System. Immunity 44,739-754 (2016)). The contiguous structure of circRNA may confer it withresistance to endosomal nucleases, resulting in evasion of thesedetectors. In this case, endosomal nucleases would be expected to becomposed primarily of exonucleases, as the presence of endonucleaseswould be expected to lead to circRNA degradation. Consistent with thispostulation, the addition of one of the two cooperative TLR8 ligands,uridine, to the media of TLR8 reporter cells was able to partiallyabrogate the immunoevasive properties of circRNA, suggesting that a lackof degradation products and therefore nuclease resistance may indeed beresponsible for TLR8 evasion by circRNA. However, no degradation producthas yet to be defined as a ligand for TLR3, which circRNA also appearsto evade in the context of TLR3-overexpressing 293 cells despitecontaining the same dsRNA motifs as the TLR3-activating linearizedcircRNA. It may be possible that RNA degradation products bind to TLR3at the dimerization interface in a similar manner to TLR8 (Roers, A.,Hiller, B. & Hornung, V. Recognition of Endogenous Nucleic Acids by theInnate Immune System. Immunity 44, 739-754 (2016)). Differences in TLR3activation by linearized circRNA was also observed, with in vitrotranscribed linearized circRNA eliciting a TLR3-mediated response whilelinearized circRNA produced by degrading purified circRNA did not (FIGS.10B and F). It is possible that degradation of circRNA by either heat orRNase H disrupts dsRNA structures required for robust TLR3 activation.

Intra-adipose injection of circRNA complexed with transfection reagentor within LNPs yielded hEpo expression that was more stable than thatfrom m1ψ-mRNA or 5moU-mRNA (FIG. 11B and FIG. 12F). However, althoughhEpo production from circRNA was observed to be close to twofold higherthan that from equimolar transfection of m1ψ-mRNA or 5moU-mRNA in 293cells at 24 hours, hEpo expression from circRNA in vivo was relativelydiminished. Several factors may have led to this result. The CVB3 IRESwas originally selected for use in circRNA based on its ability to drivetranslation in human cell lines (Wesselhoeft, R. A., Kowalski, P. S. &Anderson, D. G. Engineering circular RNA for potent and stabletranslation in eukaryotic cells. Nat. Commun. 9, 2629 (2018)). Mouseadipose or liver tissue may therefore not be the ideal cell type forCVB3 IRES-mediated translation. IRES sequences must also compete fortranslation initiation factors with endogenous transcripts bearing m⁷Gcaps. Accordingly, circRNA using viral IRES sequences to initiatetranslation could be more effective in cells with higher initiationfactor density relative to transcript density. A comprehensivecharacterization of the ability of other IRES sequences to drivetranslation from circRNA in diverse tissues is needed.

Protein expression stability from circRNA delivered intravenously by LNPto liver was not enhanced compared to that from 5moU-mRNA, although therelative magnitude of expression from circRNA at 6 hours was comparableto that obtained from adipose tissue (FIG. 12E and FIG. 16E). Thisresult highlights tissue specific stability that may be dependent onseveral factors, including general RNA turnover rate or endonucleaseactivity, sequence specific translation inhibition or degradation, andthe presence or absence of RNA stabilizing proteins. Assessment andalteration of miRNA binding sites within circRNA or depletion ofsequence-specific degradation motifs may further enhance circRNAstability and expression in select tissues.

An increase in serum cytokines was detected in mice injected withunpurified splicing reactions, but such a response in mice injected withunmodified mRNA, m1ψ-mRNA/5moU-mRNA, or circRNA was not detected.Consistent with in vitro results, a rapid decrease in hEpo expressionupon injection of unmodified mRNA and unpurified splicing reactions wasobserved, while serum hEpo after injection of m1ψ-mRNA/5moU-mRNA andcircRNA remained relatively stable, indicating that m1ψ-mRNA/5moU-mRNAand circRNA did not provoke a substantial immune response that wouldlead to RNA degradation in vivo. Formulation of circRNA into LNPs didnot alter immune sensor interactions, and analysis of serum cytokinesand local pro-inflammatory transcript levels after LNP-RNA injectionsdid not reveal an immune response against LNP-delivered circRNA.

It is believed that the enhanced expression stability of circRNA in sometissues and the ability of circRNA to avoid immune sensors without theneed for nucleoside modifications demonstrates the potential of circRNAas a vector for the expression of therapeutic proteins.

Methods:

RNA Design, Synthesis, and Purification

Linear mRNA or circRNA precursors were synthesized by runoff in-vitrotranscription from a linearized plasmid DNA template using a T7 HighYield RNA Synthesis Kit (New England Biolabs (NEB)) with the completereplacement of uridine with N1-methylpseudouridine (TrilinkBiotechnologies) for modified linear or circular RNA. After in vitrotranscription, reactions were treated with DNase I (NEB) for 15 minutes.After DNase treatment, RNA was column purified using a MEGAclearTranscription Clean-up kit (Ambion). RNA was then heated to 70° C. for 3minutes and immediately placed on ice for 2 minutes, after which linearRNA was capped using mRNA cap-2′-O-methyltransferase (NEB) and Vacciniacapping enzyme (NEB) according to the manufacturer's instructions.Polyadenosine tails were added to capped linear transcripts using E.coli PolyA Polymerase (NEB) according to manufacturer's instructions,and fully processed mRNA was column purified. For circRNA, GTP was addedto a final concentration of 2 mM along with a buffer including magnesium(50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5; NEB), and then reactionswere heated at 55° C. for 8 minutes. RNA was then column purified. Insome cases, circRNA was digested with RNase R: 20 μg of RNA was dilutedin water (86 uL final volume) and then heated at 70° C. for 3 minutesand cooled on ice for 2 minutes. 20 U RNase R and 10 uL of 10× RNase Rbuffer (Applied Biological Materials) was added, and the reaction wasincubated at 37° C. for 15 minutes; an additional 10 U RNase R was addedhalfway through the reaction. RNase R-digested RNA was column purified.In some cases, RNA was treated with a phosphatase (CIP, NEB): 20 ug ofRNA was diluted, heated and cooled as described above and then Cutsmartbuffer (NEB) was added to a final concentration of 1× along with 20 U ofCIP. The reaction was incubated at 37° C. for 15 minutes.Phosphatase-treated RNA was column purified. RNA was diluted in 50%formamide, denatured at 70° C. for 3 minutes, and then cooled to roomtemperature. RNA was then separated on precast 2% E-gel EX agarose gels(Invitrogen) on the E-gel iBase (Invitrogen) using the E-gel EX 1-2%program; ssRNA Ladder (NEB) was used as a standard. Bands werevisualized using blue light transillumination and quantified usingImageJ. For high-performance liquid chromatography, 30 μg of RNA washeated at 65° C. for 3 minutes and then placed on ice for 2 minutes. RNAwas run through a 4.6×300 mm size-exclusion column with particle size of5 μm and pore size of 2000 Å (Sepax Technologies; part number:215980P-4630) on an Agilent 1100 Series HPLC (Agilent). RNA was run inRNase-free TE buffer (10 mM Tris, 1 mM EDTA, pH:6) at a flow rate of 0.3mL/minute. RNA was detected by UV absorbance at 260 nm, but wascollected without UV detection. Resulting RNA fractions wereprecipitated with 5M ammonium acetate, resuspended in water, and then insome cases subjected to further enzymatic treatment as described above.5moU-modified Firefly Luciferase and hEpo mRNA was obtained from TrilinkBiotechnologies.

Splint Ligation

Linear precursors for splint-mediated ligation were designed to have allof the same sequence features as PIE-circularized circRNA except for theaddition of short adapter sequences onto the 5′ and 3′ ends of theprecursor RNA. These adapter sequences shared homology with the splintsused for circularization (Optimized splint:5′-GTTTGTGGTTCGTGCGTCTCCGTGCTGTTCTGTTGGTGTGGG-3′ (SEQ ID NO: 33). Splintligation precursor RNA was synthesized as described previously, except a10-fold excess of GMP was added to in vitro transcription reactions. 25ug of purified precursor RNA was heated to 70° C. for 5 minutes in thepresence of DNA splint at a concentration of 5 uM in a 90 uL reaction.The reaction was allowed to cool to room temperature, and then T4 RNALigase I Buffer (NEB) was added to a final concentration of 1×. ATP wasadded to a final concentration of 1 mM. 50 U of T4 RNA Ligase I (NEB)was added. Reactions were incubated at 37° C. for 30 minutes and thencolumn purified.

RNase H Nicking

Splicing reactions enriched for circRNA with RNase R and then columnpurified, or purified by HPLC, were heated at 70° C. for 5 minutes inthe presence of a DNA probe (5′-TTGAACCCAGGAATCTCAGG-3′(SEQ ID NO: 34))at five-fold molar excess, and then annealed at room temperature.Reactions were treated with RNase H (New England Biolabs) in theprovided reaction buffer for 15 minutes at 37° C. RNA was columnpurified after digestion.

Tissue Culture, Transfections, and Cell Viability

293 and A549 cells RAW264.7 cells (ATCC) and HEK-Blue mouse TLR3, mouseTLR7, human TLR8, Null1, and Null2 cells (Invivogen) were cultured at37° C. and 5% CO2 in Dulbecco's Modified Eagle's Medium (4500 mg/Lglucose) supplemented with 10% heat-inactivated fetal bovine serum(hiFBS, Gibco) and penicillin/streptomycin. HEK293 and HeLa cells testednegative for mycoplasma. Cells were passaged every 2-3 days. For 293 andA549 cells, 40 ng of RNA was reverse transfected into 10,000 cells/100uL per well of a 96-well plate using Lipofectamine MessengerMax(Invitrogen) according to the manufacturer's instructions. For HEK-Bluecells, 100 ng of RNA was reverse transfected into 40,000 cells/100 uLper well of a 96-well plate using Lipofectamine MessengerMax. For A549cells transfected prior to RNA harvest and qPCR, 200 ng of RNA wasreverse transfected into 100,000 cells per well of a 24-well plate usingLipofectamine MessengerMax. For experiments wherein protein expressionwas assessed at multiple time points, media was fully removed andreplaced at each time point. For experiments wherein SEAP activity orcytokines were analyzed, media was not replaced between transfection andassessment. For all transfection experiments, RNA was heated to 70° C.for 3 minutes and immediately placed on ice for 2 minutes prior tocomplexation with transfection reagent. Cell viability 36-72 hours aftertransfection was assessed using a MultiTox kit (Promega). To detect SEAPsecretion by TLR reporter and null cells, media was harvested 36-48hours after transfection and combined with HEK-Blue Detection reagent(Invivogen) to a final concentration of 1×. Media and detection reagentwere incubated overnight at 37° C. and then absorbance at 640 nm wasmeasured on an Infinite 200Pro Microplate Reader (Tecan). R848, polyI:C,and 3p-hpRNA were obtained from Invivogen.

Protein Expression Analysis

For luminescence assays, media was harvested 24 hours post-transfection.To detect luminescence from Gaussia luciferase, 20 uL of tissue culturemedium was transferred to a flat-bottomed white-walled plate (Corning).25 uL of BioLux Gaussia Luciferase reagent including stabilizer (NewEngland Biolabs) was added to each sample and luminescence was measuredon an Infinite 200Pro Microplate Reader (Tecan) after 45 seconds. Humanerythropoietin was detected by solid phase sandwich ELISA (R&D Systems)essentially according to the manufacturer's instructions. Cytokines inFIGS. 1, 3 and 5 were detected by Fireplex immunoassay (Abcam).Cytokines in FIGS. 2 and 6 were detected by individual or multipleximmunoassay (Eve Technologies).

Reverse Transcription and qPCR

Cells were washed and RNA was harvested and purified 24 hours aftertransfection using an RNeasy Mini Plus kit (Qiagen) or RNeasy Lipid Kit(Qiagen) for RNA extracted from mouse adipose tissue according to themanufacturer's instructions. Synthesis of first-strand cDNA from totalRNA was performed with High-Capacity cDNA Reverse Transcription Kitusing random hexamers (Thermo Fisher Scientific). Gene specific TaqManprimers were purchased as Assay-on-Demand (Thermo Fisher Scientific);human primers: GAPDH (Hs99999905_m1), DDX58 (Hs01061436_m1), IFN-β1(Hs01077958_s1); mouse primers: Gapdh (Mm99999915_g1), Ddx58(Mm01216853_m1), 11-6 (Mm00446190_m1), Tnf (Mm00443258_m1). The qPCRreaction was carried out using LightCycler 480 Probe Master Mix (Roche)and LightCycler 480 instrument (Roche). For each sample, threshold cyclevalues (Ct) were processed according to the comparative Ct method. Geneexpression levels were normalized to the expression of the housekeepinggene GAPDH.

Animal Experiments

All animal experiments were performed under the guidelines of the MITAnimal Care and Use Committee. 30-35 g C57Bl/6 female mice randomlyassigned to treatment or control groups were injected into visceral fatthrough the lower right mammary fat pad and peritoneum with 350 ng ofRNA complexed with MessengerMax or 1.5 picomoles of LNP-RNA in a totalvolume of 50 μL, or intravenously by tail vein injection with 0.1 mg/kgLNP-RNA. Blood samples were collected via tail bleed or cardiac punctureinto BD Microtainer tubes at the indicated time points. To collect theserum, blood was allowed to coagulate for 15-30 min and was subsequentlycentrifuged at 2000×g for 5 min at room temperature. Humanerythropoietin in 2 uL of serum was detected as described previously. Tocollect adipose tissue, mice were sacrificed and the entire lowervisceral adipose tissue was removed and frozen in liquid nitrogen forsubsequent RNA isolation.

Lipid Nanoparticle Formulation

LNPs were prepared by mixing ethanol and aqueous phase at a 1:3volumetric ratio in a microfluidic device, using syringe pumps aspreviously described. In brief, ethanol phase was prepared bysolubilizing a mixture of ionizable lipidoid cKK-E12,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Avanti),cholesterol (Sigma), and1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000](ammonium salt) (C14-PEG 2000, Avanti) at a molar ratio of35:16:46.5:2.5. The aqueous phase was prepared in 10 mM citrate buffer(pH 3) with linear mRNA or circRNA. LNPs were dialyzed against PBS in aSlide-A-Lyzer™ G2 Dialysis Cassettes, 20,000 MWCO (Thermo Fisher) for 2h at RT. The concentration of mRNA encapsulated into LNPs nanoparticleswas analyzed using Quant-iT RiboGreen assay (Thermo Fisher) according tothe manufacturer's protocol. The efficiency of mRNA encapsulation intoLNPs was calculated by comparing measurements in the absence andpresence of 1% (v/v) Triton X-100. Nanoparticle size, polydispersity(PDI), and ζ-potential were analyzed by dynamic light scattering (DLS)using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). LNPhydrodynamic diameters are reported in the volume weighting mode and arean average of three independent measurements.

CRYO-TEM

For Cryogenic Transmission Electron Microscopy (Cryo-TEM) samples wereprepared on a Gatan Cryo Plunge III (Cp3). Briefly, 3 uL of the samplewas dropped on a lacey copper grid coated with a continuous carbon filmand frozen in liquid ethane. Subsequently the frozen grid was mounted ona Gatan 626 single tilt cryo-holder. Imaging was performed using JEOL2100 FEG microscope operating at 200 kV with a magnification of10,000-60,000. All Images were recorded under low-dose conditions with aGatan 2kx2k UltraScan CCD camera.

Data Analysis and Statistics

For TLR data in FIGS. 10 and 12, absorbance measured in TLR reportercells was normalized to absorbance measured in null reporter cellscontaining only the plasmid with SEAP under the control of the IFN-βminimal promoter fused to five NF-κB and AP-1 binding sites for mTLR3and hTLR8, or null reporter cells containing only the plasmid with SEAPunder the control of the IL-12p40 minimal promoter fused to five NF-κBand AP-1 binding sites for mTLR7 (Invivogen). For all multi-day GLuc andhEpo data, expression is presented relative to the first day ofexpression for each condition. Statistical analysis of the results wasperformed by a two-tailed unpaired Welch's t-test, assuming unequalvariances. Differences were considered significant when p<0.05. For allstudies, data presented is representative of one independent experiment.

STAR Methods:

REAGENT or RESOURCE SOURCE IDENTIFIERChemicals, Peptides, and Recombinant Proteins RNase R Applied Cat#E049Biological Materials 3p-hpRNA Invivogen Cat#tlrl-hprna polyI:C InvivogenCat·tlrl-pic RNase H New England Cat#M0297S Biolabs T4 RNA Ligase 1New England Cat#M0204S BiolabsLipofectamine ™ MessengerMAXT ™ ransfection Reagent ThermoFisherCat#LMRNA003 Scientific N1-Methylpseudouridine-5′-Triphosphate TrilinkCat#N-1081 Biotechnologies CleanCap ™ EPO mRNA (5moU) Trilink Cat#L-7209Biotechnologies CleanCap ™ FLuc mRNA (5moU) Trilink Cat#L-7202Biotechnologies Alkaline Phosphatase, Calf Intestinal (CIP) New EnglandCat#M0290S Biolabs Vaccinia Capping System New England Cat#M2080SBiolabs mRNA Cap 2′-O-Methyltransferase New England Cat#M0366S BiolabsE. coli Poly(A) Polymerase New England Cat#M0276S Biolabs UridineMillipore Sigma Cat#U6381 Cytidine Millipore Sigma Cat#C46541,2-dioleoyl-sn-glycero-3-phosphoethanolamine Avanti Polar Cat#850725PLipids 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- Avanti PolarCat#700100P [methoxy-(polyethyleneglycol)-2000] LipidsN6-Methyladenosine-5′-Triphosphate Trilink Cat#N-1013 BiotechnologiesCritical Commercial Assays EGel ™ EX Agarose Gels, 2% ThermoFisherCat#G401002 Scientific HiScribe ™ T7 High Yield RNA Synthesis KitNew England Cat#E2040S BiolabsMultiTox-Fluor Multiplex Cytotoxicity Assay Promega Cat#G9200RNeasy Mini Kit Qiagen Cat#74104Human Erythropoietin Quantikine IVD ELISA Kit R & D Systems Cat#DEP00BioLux ® Gaussia Luciferase Assay Kit New England Cat#E3300 BiolabshCCL5 TaqMan ® Gene Expression Assay ThermoFisher Cat#Hs00982282m1Scientific hIFNB1 TaqMan ® Gene Expression Assay ThermoFisherCat#Hs01077958s1 Scientific hGAPDH TaqMan ® Gene Expression AssayThermoFisher Cat#Hs99999905m1 ScientifichDDX58 TaqMan ® Gene Expression Assay ThermoFisher Cat#Hs01061436m1Scientific hIL6 TaqMan ® Gene Expression Assay ThermoFisherCat#Hs00714131m1 Scientific mCcl5 TaqMan ® Gene Expression AssayThermoFisher Cat#Mm01302427m1 ScientificmIfnB1 TaqMan ® Gene Expression Assay ThermoFisher Cat#Mm00439552s1Scientific mGapdh TaqMan ® Gene Expression Assay ThermoFisherCat#Mm99999915g1 Scientific mDdx58 TaqMan ® Gene Expression AssayThermoFisher Cat#Mm01216853m1 ScientificmI16 TaqMan ® Gene Expression Assay ThermoFisher Cat#Mm00446190m1Scientific Tnf TaqMan ® Gene Expression Assay ThermoFisherCat#Mm00443258_ml Scientific Experimental Models: Cell LinesHEK-Blue ™ Null1 Cells Invivogen Cat#hkb-null1 HEKBlue ™ Null2 cellsInvivogen Cat#hkb-null2 HEK-Blue ™ mTLR3 Invivogen Cat#hkb-mtlr3HEK-Blue ™ mTLR7 Invivogen Cat#hkb-mtlr7 HEK-Blue ™ hTLR8 InvivogenCat#hkb-htlr8 293 [HEK-293] ATCC Cat#CRL-1573 A549 ATCC Cat#CCL-185RAW264,7 ATCC Cat#TIB-71 HeLa ATCC Cat#CCL-2Experimental Models: Organisms/Strains C57BL/6 Mice Charles RiverCat#C57BL/6NCr1 OligonucleotidesRNase H Probe: TTGAACCCAGGAATCTCAGG (SEQ ID NO. Described N/A 34) hereinLigation Splint: Described N/A GTTTGTGGTTCGTGCGTCTCCGTGCTGTTCTGTTGGTGTGGherein G (SEQ ID NO. 33) Recombinant DNA Plasmid: GLuc APIE CVB3 pACWesselhoeft et N/A al., 2017 Plasmid: hEpo APIE CVB3 pAC Wesselhoeft etN/A al., 2017 Plasmid: EGFP APIE EMCV Wesselhoeft et N/A al., 2017Plasmid: GLuc APIE ΔIRES Described N/A hereinPlasmid: GLuc APIE CVB3 pAC dS Wesselhoeft et N/A al., 2017Plasmid: GLuc APIE CVB3 pAC dI Wesselhoeft et N/A al., 2017Plasmid: splintGLuc CVB3 Described N/A herein Plasmid: splinthEpo CVB3Described N/A herein Plasmid: GLuc L Described N/A hereinPlasmid: hEpo L Described N/A herein

TABLE 1 SEQ ID GLucGGGAGACCCTCGAGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAG NO. 1T4PIE ACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATEMCV TCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCACCCCCCTC(Full)TCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAACAGAGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTAATTCCATTTATCAGATTTCTAG SEQ ID WeakGGGAGACCCTCGAGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGGTCAAGTGA NO. 2homologyCTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCarms 3′ AGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATG Intron SEQ IDWeak TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTNO. 3 homology AGACAATCCCGTGCTAAATTGTAGGACTAATTCCATTTATCAGATTTCTAGarms 5′ Intron SEQ ID StrongGGGAGACCCTCGAATGGAATTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGG NO. 4homologyTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGarms 3′ TGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGIntron SEQ ID StrongTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGT NO. 5homology AGACAATCCCGTGCTAAATTGTAGGACTAATTCCATTTATCAGATTTCTAG arms 5′Intron SEQ ID T4 ACTGCAAGTTGTCTATCGTTACGGTAAGTCACCTTATTTCA NO. 6Disruptive spacer SEQ ID T41 5′ GGTAGTGGTGCTACTAACTTCAGCCTGCTGAAGCANO. 7 Permissive spacer 1 SEQ ID T42 5′GGTAGTAAACTACTAACTACAACCTGCTGAAGCA NO. 8 Permissive spacer 2 SEQ ID2400 ntGGGAGACCCTCGAATGGAATTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGG NO. 9(Full) TCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCAGGTAGTAAACTACTAACTACAACCTGCTGAAGCACCCCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAACAGAGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTAATTCCATTTATCAGATTTCTAG SEQ ID 4800 ntGGGAGACCCTCGAATGGAATTGGTTCTACATAAATGCCTAACGACTATCCCTTTGGGGAGTAGGG NO. 10(Full) TCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATAATGCTACCGTTTAATATTGCGTCAGGTAGTAAACTACTAACTACAACCTGCTGAAGCACCCCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTAGGTCTCATATCACCTTGGCTTTCGCCGTATTCACATGCTGGAACACATCATGCTAGCTTTAACATCGGGGAGTTACGATCCGTGAAAAGACGGATATATTGCCCTTGTATAGGACTATATTCCGGAGGGATTAGAATTTATAGTTGGAGAGCTTCATACCCCACTGAGCTTTTCACTGTATGCTAGTAATTATACTTCATTTGCTCGTTGGGTAGATGCGTTCTTCTCGCACAAGCCGATGATTTCCGAGTTTCTTTTCAGCGGCCGTAAAGCCTCGACACGTGACGCACTGTGAACCGCACCCGTATACCTAATAGAGGCAGTTAACTTCATTCAGCCACATAAGGGGTGATAACACCGACTGCCCAAGTACGGAATTAAGAAAATGGATAATGAAGATTATGAGATCACCGCTACATTAGCAACGCTTGGTGCTTTAATTGGCATGTATGACCAATCTACAACCTGGGGGGAGGGTACCTCTTTGAGATGTACGATGCAGCCTAAAGGGTAACGTTATGCAAGTGGTCAAGAGCCTAGCATGCTTATGCGGTTTATCAAAATGTATCGCACTTTATGCTAGGTAATGTGTGTTCTCCACGGTATCCATAAGCTTGCCTAAATACTGAAGTCTACGAGAAAACTATGGGATATTTGTGCATATATTACCCATAGTTATCCTGGAGCAGTCCGTTCCCACGTAGAATGTAGCGAATTGCGTGGCTGGCTTCAAACATAGCACCGAACAGTAGATCTAGTTGCGCCCCTTCCAAGTTTACAGTTAGGTAAACCTTCACGATAGAAAGTTGGGAACAAGGCCGCATTCAACCTTTACGATCACTTCCAGAAAGGGATTGTGGGTAGGAGACACCACGCCCTCAGATCACGTCGATCACTTGTTATAAGGTCAAATGTGAGAAACGCGTCAGAAGGGAGTTGGTGCTTGCTTATTTCTTTTCCAGACTCGTCGTTGGATCAACCTATCTCATGACCCTAGCTCTAGTATGTCTGGTGGTAAAGGAGCTGCGCTGGATGCATTTATTCTGCTGGGAGAAATAATCGCGGATATTATCCTTTTTCAAAGAGACGCCGAACTAATGACTTGTCGAGAGGAATCGGCATGGTTTCGTACCTTGCCAGCATTCCCAATTTTTTTTTATTTGCTTGGGTCTTATAAAGGAAATCGACAATTTGGGTAAAATGGTGCAAAGAATCTACCCGTTGGAATATTTTACTGGAGTCACCGGGGGAGCTTCGAGGACACACCTACCTGGTCTAACCCAGCCTACTTGTAAGATATGTTAACGTCGGCACCGTCATTGTAGTTATCTTATTTAAGGCGACACGAGACGTGAGAACTTTTGCATTGCATATGTAACGGTCAATGTCGTACATGCGACACCATTGGATCGCTACCGTAAAAGTACACGTTACGGGGGTAGCTGGTGTACCTAAGCGCGACCCGGAACACCTACACCCGCTAGTTTAGCTTGTGAAAGTGCGGCGCTGCCTGTGATTCACGCGTTGTATGGACAACGTTGTACCATTCGTAGCAGACTTTGATCAATGATGTAGTTATGCCATGCCCGAAACAAACTATAGACATTTTCGAAAACGTTCCACTGAGTTAATCCTTAAGCCATGCAATTTTATGAAAATTTATTAGGCTAGCGGAAATTACGTTCCAAGTTCTGGAACCCTTATATCGATCAAGGCTGCAGACCTAATGGCTTGTGTTCCTGAAACATGTTACGTTGCCATTAACTCGGGAGTCGAGTACGTGCCATGTGTTGTGATGGGAGGTACTCGTTTGCGGAAAGGCATCTGCCCAAAAACACATTAGGTCATTAACGTCCCGTTACGGTAGATATGGCCACGGTCCACATAAACCGCTCATGGGTAAAAAGGATTCCTATACCTAACGGCTAGATGGCCAGGTATGTGCAATTTGGGCAGGATCCCGTTGGACGTGACATCTCAATGGCCTGAGAGGTCTGAGACCCCCGATGGAGATAGTTTAATCAAACTTTTGAAATGCCAAGGCACAGCTAGATTTAGATAGTCAACGCCATCGACTTTGCATTTTCGACATATACTCTTGCCATTATGAGAGTGACGCGGATAAGAGGTAGGGATGCATGAGTAAAAGAGAGCGGTTTTACGTTCAATATGTGGAAGGATGCTCTAGCCGGGAGTGAGGACACTAAACGCTTGTCATGCACAGTTACTGTGCGGCGTATTGTTAGGGATGCGGTTGTAGTAGTCAAACGGCCAGAAAATGTGTCTCATTTTGAATTCGCGATCTCAGATCTCCGTGAAATGATCTTCGGAATTCAACTCTCATCGGGACAGCAGGACGCGTGCTAACTTAGGGCGTTTCAACTGTGATCCGAATACGTATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAACAGAGATGTTTTCTTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGTAGACAATCCCGTGCTAAATTGTAGGACTAATTCCATTTATCAGATTTCTAG SEQ ID Ana1.0GGGAGACCCTCGACCGTCGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGT NO. 11(Full) GCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCAGAAACCAACTTTATTACTATATTCCCCACAACCCCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGACCAGTGGACAATCGACGGATAACAGCATATC TAGSEQ ID Ana2.0GGGAGACCCTCGACCGTCGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGT NO. 12(Full) GCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCATGATCTGAAACCAACTTTATTACTATATTCCCCACAACCCCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGACCAGTGGACAATCGACGGATAACAGCATATCTAG SEQ ID Ana3.0GGGAGACCCTCGACCGTCGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGT NO. 13(Full) GCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTCCCTCCACCCCCACGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAAACCCCCCTCTCCCTCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAAAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCGAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGACCAGTGGACAATCGACGGATAACAGCATATCTAG SEQ ID hEpoATGGGAGTGCATGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCACTGCTGTCTCTCCCTCTGGGCNO. 14 CTCCCAGTGCTGGGCGCACCACCAAGACTCATCTGTGACAGCAGAGTGCTGGAGAGGTATCTCTTGGAGGCCAAGGAGGCTGAGAACATTACCACAGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTTGGGCAACAAGCAGTTGAAGTGTGGCAAGGCCTGGCCCTGCTGTCTGAAGCTGTCCTGAGGGGCCAGGCACTGTTGGTCAACTCTTCCCAGCCTTGGGAGCCCCTGCAACTGCATGTGGATAAAGCAGTGAGTGGCCTTAGAAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCACAGAAGGAAGCCATCTCCCCTCCAGATGCAGCCTCAGCAGCTCCACTCAGAACAATTACTGCTGACACTTTTAGAAAACTCTTTAGGGTGTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGTGAGGCATGTAGGACAGGGGACAGATAA SEQ IDEGFP ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGNO. 15 ACGTAAACGGCCACAAGTTCAGCGTGTCTGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA SEQ ID FLucATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCG NO. 16CTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGGGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGTGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGTGTAA SEQ ID Cas9ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTAC NO. 17AGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAA SEQ ID sgGFPGGGCGAGGAGCGCACCGGGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG NO. 18UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU SEQ ID RNase HCATGGTTGTGGCCATATTATCATCG NO. 19 Probe SEQ ID SpliceCGATCGTCGACATTCCTGAG NO. 20 Junction PCR F SEQ ID SpliceATGCTCGTCAAGAAGACAGG NO. 21 Junction PCR R SEQ ID ABPVTTTGGGAATCGCAACACAACATGGTTACCCATAGATTGAGGAAATTTCCAATAAACTCAATCTTA NO. 22AGGCTTGTTGTGTTGGACAAGGTGCCCTATTTAGGGTGAGGAGCCTTGCTGGCAGCCCCAGTGAATCCTCTATTGGATAGGAACAGCTATATTGGGTAGTTGTAGCAGTTGTATTCAAACGAATGCAGCGTTCCGAAATACCATACCT SEQ ID CSFVGTATACGAGGTTAGTTCATTCTCGTATACACGATTGGACAAATCAAAATTATAATTTGGTTCAGG NO. 23GCCTCCCTCCAGCGACGGCCGAACTGGGCTAGCCATGCCCATAGTAGGACTAGCAAACGGAGGGACTAGCCGTAGTGGCGAGCTCCCTGGGTGGTCTAAGTCCTGAGTACAGGACAGTCGTCAGTAGTTCGACGTGAGCAGAAGCCCACCTCGAGATGCTACGTGGACGAGGGCATGCCCAAGACACACCTTAACCCTAGCGGGGGTCGCTAGGGTGAAATCACACCACGTGATGGGAGTACGACCTGATAGGGCGCTGCAGAGGCCCACTATTAGGCTAGTATAAAAATCTCTGCTGTACATGGCAC SEQ ID CVB3TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGG NO. 24TACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAA SEQ ID EMCV2TTGCCAGTCTGCTCGATATCGCAGGCTGGGTCCGTGACTACCCACTCCCCCTTTCAACGTGAAGG NO. 25CTACGATAGTGCCAGGGCGGGTACTGCCGTAAGTGCCACCCCAAACAACAACAACAAAACAAACTCCCCCTCCCCCCCCTTACTATACTGGCCGAAGCCACTTGGAATAAGGCCGGTGTGCGTTTGTCTACATGCTATTTTCTACCGCATTACCGTCTTATGGTAATGTGAGGGTCCAGAACCTGACCCTGTCTTCTTGACGAACACTCCTAGGGGTCTTTCCCCTCTCGACAAAGGAGTGTAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTAAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGTGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACGTGCTTTACACGTGTTGAGTCGAGGTGAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAACCACGATTACAAT SEQ ID EV71TTAAAACAGCTGTGGGTTGTCACCCACCCACAGGGTCCACTGGGCGCTAGTACACTGGTATCTCG NO. 26GTACCTTTGTACGCCTGTTTTATACCCCCTCCCTGATTTGCAACTTAGAAGCAACGCAAACCAGATCAATAGTAGGTGTGACATACCAGTCGCATCTTGATCAAGCACTTCTGTATCCCCGGACCGAGTATCAATAGACTGTGCACACGGTTGAAGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGAGAAGCCTAGTAACGCCATTGAAGTTGCAGAGTGTTTCGCTCAGCACTCCCCCCGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCTATGGGGTAACCCATAGGACGCTCTAATACGGACATGGCGTGAAGAGTCTATTGAGCTAGTTAGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATACCCTTAATCCAAAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCTTTTTATTCTTGTATTGGCTGCTTATGGTGACAATTAAAGAATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGTCAAACAGAGCTATTGTATATCTCTTTGTTGGATTCACACCTCTCACTCTTGAAACGTTACACACCCTCAATTACATTATACTGCTGAACACGAAGCG SEQ ID HAVTTCAAGAGGGGTTTCCGGAGTTTTCCGGAGCCCCTCTTGGAAGTCCATGGTGAGGGGACTTGATA NO. 27CCTCACCGCCGTTTGCCTAGGCTATAGGCTAAATTTCCCTTTCCCTGTCCTTCCCTTATTTCCCTTTATCTTGTTTGTAAATATTAATTCCTGCAGGTTCAGGGTTCTTTAATCTGTTTCTCTATAAGAACACTCAATTTTCACGCTTTCTGTCTTCTTTCTTCCAGGGCTCTCCCCTTGCCCTAGGCTCTGGCCGTTGCGCCCGGCGGGGTCAACTCCATGATTAGCATGGAGCTGTAGGAGTCTAAATTGGGGACGCAGATGTTTGGGACGTCGCCTTGCAGTGTTAACTTGGCTCTCATGAACCTCTTTGATCTTTCACAAGGGGTAGGCTACGGGTGAAACCCCTTAGGCTAATACTTCTATGAAGAGATGCCTTGGATAGGGTAACAGCGGCGGATATTGGTGAGTTGTTAAGACAAAAACCATTCAACGCCGGAGGACTGGCTCTCATCCAGTGGATGCATTGAGTGAATTGATTGTCAGGGCTGTCTTTAGGTTTAATCTCAGACCTCTCTGTGCTTAGGGCAAACACTATTTGGCCTTAAATGGGATCCTGTGAGAGGGGGTCCCTCCATTGACAGCTGGACTGTTCTTTGGGGCCTTATGTAGTGTTTGCCTCTGAGGTACTCAGGGGCATTTAGGTTTTTCCTCACTCTTAAACAATA SEQ ID HRV2TTAAAACTGGATCCAGGTTGTTCCCACCTGGATTTCCCACAGGGAGTGGTACTCTGTTATTACGGTNO. 28AACTTTGTACGCCAGTTTTATCTCCCTTCCCCCATGTAACTTAGAAGTTTTTCACAAAGACCAATAGCCGGTAATCAGCCAGATTACTGAAGGTCAAGCACTTCTGTTTCCCCGGTCAATGTTGATATGCTCCAACAGGGCAAAAACAACTGCGATCGTTAACCGCAAAGCGCCTACGCAAAGCTTAGTAGCATCTTTGAAATCGTTTGGCTGGTCGATCCGCCATTTCCCCTGGTAGACCTGGCAGATGAGGCTAGAAATACCCCACTGGCGACAGTGTTCTAGCCTGCGTGGCTGCCTGCACACCCTATGGGTGTGAAGCCAAACAATGGACAAGGTGTGAAGAGCCCCGTGTGCTCGCTTTGAGTCCTCCGGCCCCTGAATGTGGCTAACCTTAACCCTGCAGCTAGAGCACGTAACCCAATGTGTATCTAGTCGTAATGAGCAATTGCGGGATGGGACCAACTACTTTGGGTGTCCGTGTTTCACTTTTTCCTTTATATTTGCTTATGGTGACAATATATACAATATATATATTGGCACCATGG SEQ ID HTLVGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTC NO. 29GCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAGAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGTTCAACTCTGCGTCTTTGTTTCGTTTTCTGTTCTGCGCCGCTACAGATCGAAAGTTCCACCCCTTTCCCTTTCATTCACGACTGACTGCCGGCTTGGCCCACGGCCAAGTACCGGCGACTCCGTTGGCTCGGAGCCAGCGACAGCCCATCCTATAGCACTCTCCAGGAGAGAAACTTAGTACACAGTTGGGGGCTCGTCCGGGATACGAGCGCCCCTTTATTCCCTAGGCA SEQ ID PVTTAAAACAGCTCTGGGGTTGTACCCACCCCAGAGGCCCACGTGGCGGCTAGTACTCCGGTATTGC NO. 30GGTACCCTTGTACGCCTGTTTTATACTCCCTTCCCGTAACTTAGACGCACAAAACCAAGTTCAATAGAAGGGGGTACAAACCAGTACCACCACGAACAAGCACTTCTGTTTCCCCGGTGATGTCGTATAGACTGCTTGCGTGGTTGAAAGCGACGGATCCGTTATCCGCTTATGTACTTCGAGAAGCCCAGTACCACCTCGGAATCTTCGATGCGTTGCGCTCAGCACTCAACCCCAGAGTGTAGCTTAGGCTGATGAGTCTGGACATCCCTCACCGGTGACGGTGGTCCAGGCTGCGTTGGCGGCCTACCTATGGCTAACGCCATGGGACGCTAGTTGTGAACAAGGTGTGAAGAGCCTATTGAGCTACATAAGAATCCTCCGGCCCCTGAATGCGGCTAATCCCAACCTCGGAGCAGGTGGTCACAAACCAGTGATTGGCCTGTCGTAACGCGCAAGTCCGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTATTTTATTGTGGCTGCTTATGGTGACAATCACAGATTGTTATCATAAAGCGAATTGGATTGGCCATCCGGTGAAAGTGAGACTCATTATCTATCTGTTTGCTGGATCCGCTCCATTGAGTGTGTTTACTCTAAGTACAATTTCAACAGTTATTTCAATCAGACAATTGTATCATA SEQ ID CVB3-GGGAGACCCTCGACCGTCGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGT NO. 31GLuc- GCAGAGACTCGACGGGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAApAC AGCCAATAGGCAGTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTG(Full) GGTTCAAGTCCCTCCACCCCCACGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACATTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAAATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAAAAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCGAGACGCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCAGGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAAGACCAGTGGACAATCGACGGATAACAGCATATCTAG SEQ ID UnmodifiedGGGAGACCCTCGAATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCA NO. 32Linear AGCCCACCGAGAACAACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGAGLuc TCTCGATGCTGACCGCGGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATG(Full) GAAGCCAATGCCCGGAAAGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAAGTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCGAGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCGCACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTTCTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGGGCCGGTGGTGACTAATCTAG SEQ ID HCVgccagccccctgatgggggcgacactccaccatgaatcactcccctgtgaggaactactgtcttcacgcagaaagcNO. 35gtctagccatggcgttagtatgagtgtcgtgcagcctccaggaccccccctcccgggagagccatagtggtctgcggaaccggtgagtacaccggaattgccaggacgaccgggtcctttcttggataaacccgctcaatgcctggagatttgggcgtgcccccgcaagactgctagccgag SEQ ID EMCVcccccctctccctccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgtNO. 36tattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtatcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgc SEQ ID NRFCAGAGTAATGACATGGTTCCTTCCATCCTCCAAAGGTGACCAATAATAGTTTGTAAGTATCATTA NO. 37TGAACTAATGAATTTTCAACATATTTGATATATTTCAATCCATTGCCATCATTGTTCTTATCGATATTTGAGTTGGCTCACTTTGCCAGTAAGAGTCTATTCAAATTGGCTTCTGAGTCCATTTGACACAACA CCTSEQ ID CRPVAAAGCAAAAATGTGATCTTGCTTGTAAATACAATTTTGAGAGGTTAATAAATTACAAGTAGTGCT NO. 38ATTTTTGTATTTAGGTTAGCTATTTAGCTTTACGTTCCAGGATGCCTAGTGGCAGCCCCACAATATCCAGGAAGCCCTCTCTGCGGTTTTTCAGATTAGGTAGTCGAAAAACCTAAGAAATTTACCTGCTA CATSEQ ID GTXTTCTGACATCCGGCGGGTATTTCAGAACCGGCGGGTAGTACTGTACCGGCGGGTTTCTGACATCC NO. 39GGCGGGTTACAGTCATCCGGCGGGTTACTACAGTCCGGCGGGTTACTCAGAACCGGCGGGTTAGAATTCCTCCGGCGGGTGACTCACAACCCCAGAAACAGAGCC SEQ ID Rbm3TTTATAATTTCTTCTTCCAGAAGAATTTGTTGGTAAAGCCACC NO. 40 SEQ ID TMEVCAATCTTTGATGTCGTCTGCGGTGAATACGCTAATCGTGTTTTCACCATCCTTGGCAAAGAGAAC NO. 41GGTCTCCTGACTGTTGAACAAGCCGTGCTTGGCTTGCCGGGTATGGATCCCATGGAGAAAGACACCTCCCCTGGATTGCCCTACACCCAACAAGGACTCAGACGAACTG SEQ ID PPVCTAGGGCGCGCCAGTCCTCCAAACACTCAACACACAGACCCGGAGGCTGTCGCTTCAGGTGTGTC NO. 42ATCTATCACAGGTCCCATGTCGACATTTATGGCATCACCCACTGTTGAGGAACTTGCCGGAGACACATCAGATAGGTTGTTCCAGCTAATTGCAGGTAACTCATCCCTTATTACCCAGGAGTCAGCACGA CTSEQ ID Anal 5′ GaaaccaactttattactatattccccacaA NO. 43 SEQ ID Ana2 5′cgccggaaacgcaatagccgaaaaacaaaaaacaaaaaaA NO. 44 (internal homology)SEQ ID Ana2 3′ aaaaaacaaaaaacaaaacggctattatgcgttaccggcg NO. 45 (internalhomology) SEQ ID Ana3 5′cgccggaaacgcaatagccgaaaaacaaaaaacaaaaaaAacaaaaaaaaaaccaaaaaaacaaaacacaNO. 46 SEQ ID Ana4 5′cgccggaaacgcaatagccgaaaaacaaaaaacaaaaaaAaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaNO. 47 SEQ ID Ana5 5′ TGATCTGaaaccaactttattactatattccccacaA NO. 48(internal homology) SEQ ID Ana6 5′ GaaaccaactttattactatattcctcttaANO. 49 (PolioV) SEQ ID Ana7 5′ GaaaccaactttattactggcatatccgtccccacaANO. 50 SEQ ID Ana pA 5′ aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa NO. 51 SEQ IDAna pA 3′ aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa NO. 52 SEQ ID Ana pACacaaaaaaaaaaccaaaaaaacaaaacaca NO. 53 5′ SEQ ID Ana pT 5′ttttttttttttttttttttttttttttttttt NO. 54 SEQ ID Ana pT 3′ttttttttttttttttttttttttttttttttt NO. 55 SEQ ID Ana pC 5′ccccccccccccccccccccccccccccccccc NO. 56 SEQ ID Ana pC 3′ccccccccccccccccccccccccccccccccc NO. 57 SEQ ID Ana pG 3′ggggggggggggggggggggggggggggggggg NO. 58 SEQ ID BG 5′ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC NO. 59 SEQ ID BG 3′gctcgctttcttgctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattNO. 60 atgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgc SEQ ID5′ HHV GGACAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCNO. 61 (alsoCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCG knownTCCTTGACACG simply as ‘5′ UTR’) SEQ ID 3′ HGHCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCNO. 62 (also CACCAGCCTTGTCCTAATAAAATTAAGTTGCATCAAGCT known simply as‘3′ UTR’) SEQ ID AL 5′ ctagcttttctcttctgtcaaccccacacgcctttggcaca NO. 63SEQ ID AL 3′CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGC NO. 64TTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCG SEQ ID Ana1 5′ccgtcgattgtccactggtc NO. 65 SEQ ID Ana1 3′ gaccagtggacaatcgacgg NO. 66SEQ ID T4 5′ aatctgataaat NO. 67 inherent SEQ ID T4 3′ atttatcagattNO. 68 inherent SEQ ID T41 5′ agcctacgatcgggctaacagctcgaatctgataaatNO. 69 SEQ ID T41 3′ atttatcagattcgagctgttagcccgatcgtaggct NO. 70 SEQ IDT42 5′ GAatggaattggttctaca NO. 71 SEQ ID T42 3′ TGTAGGACTAATTCCATTTNO. 72 SEQ ID T4 5′ ggttctaca NO. 73 Weak SEQ ID T4 3′ TGTAGGACT NO. 74Weak SEQ ID Ana2 5′ ggtaactgtccgtcgattgtccactggtc NO. 75 SEQ ID Ana2 3′gaccagtggacaatcgacggacagttacc NO. 76

Wesselhoeft, R. A., et al., “RNA Circularization DiminishesImmunogenicity and Can Extend Translation Duration In Vivo,” MolecularCell, vol. 74, pages 508-520 (2019) is incorporated herein by referencein its entirety.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While example embodiments have been particularly shown and described, itwill be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe embodiments encompassed by the appended claims.

What is claimed is:
 1. A method of expressing a chimeric antigenreceptor (CAR) in a cell comprising delivering to the cell a circularRNA transcribed from a vector, wherein said vector comprises thefollowing elements: a) a 5′ homology arm, b) a 3′ Group I self-splicingintron fragment containing a 3′ splice site dinucleotide, c) a 5′ spacersequence, d) an internal ribosome entry site (IRES), e) a proteinencoding region encoding a chimeric antigen receptor, f) a 3′ spacersequence, g) a 5′ Group I self-splicing intron fragment containing a 5′splice site dinucleotide, and h) a 3′ homology arm, wherein said vectorallows the production of a circular RNA that is translatable orbiologically active inside eukaryotic cells, and wherein the circularRNA is delivered into the cell in vitro, trans-arterially,subcutaneously, intratumorally, intramedullary, intranodally,intravenously, intrathecally or intraperitoneally.
 2. The method ofclaim 1, wherein the vector further comprises an RNA polymerasepromoter.
 3. The method of claim 2, wherein the vector has a 3′ end anda 5′ end, and the RNA polymerase promoter is positioned at the 5′ end ofthe vector.
 4. The method of claim 2, wherein the RNA polymerasepromoter is a T7 virus RNA polymerase promoter, T6 virus RNA polymerasepromoter, SP6 virus RNA polymerase promoter, T3 virus RNA polymerasepromoter, or T4 virus RNA polymerase promoter.
 5. The method of claim 1,wherein at least one spacer comprises a polyA sequence.
 6. The method ofclaim 1, wherein at least one of the spacer sequences comprises apolyA-C sequence.
 7. The method of claim 1, wherein the circular RNA isdelivered into the cell intravenously.
 8. A method of expressing achimeric antigen receptor (CAR) in a cell comprising delivering to thecell a circular RNA, wherein the circular RNA comprises in the followingorder, a portion of a 3′ Group I self-splicing intron fragment, anInternal Ribosome Entry Site (IRES), a protein encoding region encodinga chimeric antigen receptor (CAR), and a portion of a 5′ Group Iself-splicing intron fragment, and wherein the circular RNA is deliveredinto the cells in vitro, trans-arterially, subcutaneously,intratumorally, intramedullary, intranodally, intravenously,intrathecally or intraperitoneally.
 9. The method of claim 8, whereinthe cell is an immune cell.
 10. The method of claim 9, wherein the cellis T cell, B cell, macrophage, or dendritic cell.
 11. The method ofclaim 9, wherein the immune cell is present in a subject in needthereof.
 12. The method of claim 8, wherein the circular RNA istransfected into the cell using lipofection or electroporation.
 13. Themethod of claim 8, wherein the circular RNA is transfected into the cellusing a delivery vehicle.
 14. The method of claim 13, wherein thedelivery vehicle comprises a nanocarrier selected from the groupconsisting of a lipid, a polymer, and a lipo-polymer hybrid.
 15. Themethod of claim 13, wherein the delivery vehicle is a lipidnanoparticle.
 16. The method of claim 8, wherein the IRES is selectedfrom an IRES sequence from a virus or a gene selected from the groupconsisting of Taura syndrome virus, Triatoma virus, Theiler'sencephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1,Rhopalosiphum padi virus, Reticuloendotheliosis virus, fuman poliovirus1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2,Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1,Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus,Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus,Human enterovirus 71, Equine rhinitis virus, Ectropis obliquapicoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila CVirus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viraldiarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus,Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscuschlorotic ringspot virus, Classical swine fever virus, Human FGF2, HumanSFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, HumanAT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc,Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc,Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1,Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx,Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A,Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiaeYAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnip crinklevirus, Coxsackievirus B3 (CVB3) and Coxsackievirus A (CVB1/2).
 17. Themethod of claim 16, wherein the IRES is a IRES sequence fromCoxsackievirus B3 (CVB3).
 18. The method of claim 16, wherein the IRESis an IRES sequence from Encephalomyocarditis virus (EMCV).
 19. Themethod of claim 8, wherein the 3′ Group I intron fragment and the 5′Group I intron fragment are from a Cyanobacterium Anabaena sp.Pre-tRNA-Leu gene.
 20. The method of claim 8, wherein the 3′ Group Iintron fragment and 5′ Group I intron fragment are from a T4 phage Tdgene.
 21. The method of claim 8, wherein the circular RNA is formed bysplint ligation of a precursor RNA.
 22. The method of claim 8, whereinthe circular RNA is circularized through ligation of the 5′ terminus ofthe nucleic acid to the 3′ terminus of the precursor RNA.
 23. The methodof claim 8, wherein circular RNA is delivered into the celltrans-arterially, subcutaneously, intratumorally, intramedullary,intranodally, intravenously, intrathecally or intraperitoneally.
 24. Themethod of claim 8, wherein the circular RNA is delivered into the cellin vitro.
 25. The method of claim 8, wherein the circular RNA comprisesa 5′ spacer sequence between the 3′ Group I intron fragment and theIRES, and a 3′ spacer sequence between the protein encoding regionencoding a chimeric antigen receptor (CAR) and the 5′ Group I intronfragment.
 26. The method of claim 25, wherein the first and secondspacers each have a length of about 10 to about 60 nucleotides.
 27. Themethod of claim 8, wherein the IRES is an IRES sequence of Salivirus.28. The method of claim 8, wherein the circular RNA is delivered intothe cell intravenously.